Ang II-induced Abdominal Aortic Aneurysm Formation Research Articles

Porphyromonas gingivalis GroEL accelerates abdominal aortic aneurysm formation by matrix metalloproteinase-2 SUMOylation in vascular smooth muscle cells: A novel finding for the activation of MMP-2.

Infection is a known cause of abdominal aortic aneurysm (AAA), and matrix metalloproteases-2 (MMP-2) secreted by vascular smooth muscle cells (SMCs) plays a key role in the structural disruption of the middle layer of the arteries during AAA progression. The periodontal pathogen Porphyromonas gingivalis is highly associated with the progression of periodontitis. GroEL protein of periodontal pathogens is an important virulence factor that can invade the body through either the bloodstream or digestive tract and is associated with numerous systemic diseases. Although P. gingivalis aggravates AAA by increasing the expression of MMP-2 in animal studies, the molecular mechanism through which P. gingivalis regulates the expression of MMP-2 is still unknown and requires further investigation. In this study, we first confirmed through animal experiments that P. gingivalis GroEL promotes MMP-2 secretion from vascular SMCs, thereby aggravating Ang II-induced aortic remodeling and AAA formation. In addition, rat vascular SMCs and A7r5 cells were used to investigate the underlying mechanisms in vitro. The results demonstrated that GroEL can promote the interaction between the K639 site of MMP-2 and SUMO-1, leading to MMP-2 SUMOylation, which inhibits the reoccurrence of non-K639-mediated monoubiquitylation. Hence, the monoubiquitylation-mediated lysosomal degradation of MMP-2 is inhibited, consequently promoting MMP-2 stability and production. SUMOylation may facilitate intra-endoplasmic reticulum (ER) and Golgi trafficking of MMP-2, thereby enhancing its transport capacity. In conclusion, this is the first report demonstrating the presence of a novel posttranslational modification, SUMOylation, in the MMP family, suggesting that P. gingivalis GroEL may exacerbate AAA formation by increasing MMP-2 production through SUMOylation in vascular SMCs. This study also provides a novel perspective on the role of SUMOylation in MMP-2-induced systemic diseases.

Pharmacological Inhibition of MMP-12 Exerts Protective Effects on Angiotensin II-Induced Abdominal Aortic Aneurysms in Apolipoprotein E-Deficient Mice.

Human abdominal aortic aneurysms (AAAs) are characterized by increased activity of matrix metalloproteinases (MMP), including MMP-12, alongside macrophage accumulation and elastin degradation, in conjunction with superimposed atherosclerosis. Previous genetic ablation studies have proposed contradictory roles for MMP-12 in AAA development. In this study, we aimed to elucidate if pharmacological inhibition of MMP-12 activity with a phosphinic peptide inhibitor protects from AAA formation and progression in angiotensin (Ang) II-infused Apoe-/- mice. Complimentary studies were conducted in a human ex vivo model of early aneurysm development. Administration of an MMP-12 inhibitor (RXP470.1) protected hypercholesterolemia Apoe-/- mice from Ang II-induced AAA formation and rupture-related death, associated with diminished medial thinning and elastin fragmentation alongside increased collagen deposition. Proteomic analyses confirmed a beneficial effect of MMP-12 inhibition on extracellular matrix remodeling proteins combined with inflammatory pathways. Furthermore, RXP470.1 treatment of mice with pre-existing AAAs exerted beneficial effects as observed through suppressed aortic dilation and rupture, medial thinning, and elastin destruction. Our findings indicate that pharmacological inhibition of MMP-12 activity retards AAA progression and improves survival in mice providing proof-of-concept evidence to motivate translational work for MMP-12 inhibitor therapy in humans.

Adenosine kinase inhibition protects mice from abdominal aortic aneurysm via epigenetic modulation of VSMC inflammation.

Abdominal aortic aneurysm (AAA) is a common, serious vascular disease with no effective pharmacological treatment. The nucleoside adenosine plays an important role in modulating vascular homeostasis, which prompted us to determine whether adenosine kinase (ADK), an adenosine metabolizing enzyme, modulates AAA formation via control of the intracellular adenosine level, and to investigate the underlying mechanisms. We used a combination of genetic and pharmacological approaches in murine models of AAA induced by calcium chloride (CaCl2) application or angiotensin II (Ang II) infusion to study the role of ADK in the development of AAA. In vitro functional assays were performed by knocking down ADK with adenovirus-short hairpin RNA in human vascular smooth muscle cells (VSMCs), and the molecular mechanisms underlying ADK function were investigated using RNA-sequencing, isotope tracing, and chromatin immunoprecipitation quantitative polymerase chain reaction (ChIP-qPCR). The heterozygous deficiency of ADK protected mice from CaCl2- and Ang II-induced AAA formation. Moreover, specific knockout of ADK in VSMCs prevented Ang II-induced AAA formation, as evidenced by reduced aortic extracellular elastin fragmentation, neovascularization, and aortic inflammation. Mechanistically, ADK knockdown in VSMCs markedly suppressed the expression of inflammatory genes associated with AAA formation, and these effects were independent of adenosine receptors. The metabolic flux and ChIP-qPCR results showed that ADK knockdown in VSMCs decreased S-adenosylmethionine (SAM)-dependent transmethylation, thereby reducing H3K4me3 binding to the promoter regions of the genes that are associated with inflammation, angiogenesis, and extracellular elastin fragmentation. Furthermore, the ADK inhibitor ABT702 protected mice from CaCl2-induced aortic inflammation, extracellular elastin fragmentation, and AAA formation. Our findings reveal a novel role for ADK inhibition in attenuating AAA via epigenetic modulation of key inflammatory genes linked to AAA pathogenesis.

Pentamethylquercetin attenuates angiotensin II-induced abdominal aortic aneurysm formation by blocking nuclear translocation of C/EBPβ at Lys253

BackgroundPentamethylquercetin (PMQ) is a natural polymethyl flavonoid that possesses anti-apoptotic and other biological properties. Abdominal aortic aneurysm (AAA), a fatal vascular disease with a high risk of rupture, is associated with phenotypic switching and apoptosis of medial vascular smooth muscle cells (VSMCs). This study aimed to investigate the protective effects of PMQ on the development of AAA and the underlying mechanism. MethodsApoE−/− mice were continuously infused with angiotensin II (Ang II) for 4 weeks to develop the AAA model. Intragastric administration of PMQ was initiated 5 days before Ang II infusion and continued for 4 weeks. In vitro, VSMCs were cultured and pretreated with PMQ, stimulated with Ang II. Real-time PCR, western blotting, and immunofluorescence staining were used to examine the roles and mechanisms of PMQ on the phenotypic switching and apoptosis of VSMCs. ResultsPMQ dose-dependently reduced the incidence of Ang II-induced AAA, aneurysm diameter enlargement, elastin degradation, VSMCs phenotypic switching and apoptosis. Furthermore, PMQ also inhibited phenotypic switching and apoptosis in Ang II-stimulated VSMCs. PMQ exerted protective effects by regulating the C/EBPβ/PTEN/AKT/GSK-3β axis. AAV-mediated overexpression of PTEN reduced the therapeutic effects of PMQ in the AAA model mice, suggesting that the effects of PMQ on Ang II-mediated AAA formation were related to the PTEN/AKT/GSK-3β axis. PMQ inhibited VSMCs phenotypic switching and apoptosis by bounding to C/EBPβ at Lys253 with hydrogen bond to regulate C/EBPβ nuclear translocation and PTEN/AKT/GSK-3β axis, thereby inhibiting Ang II-induced AAA formation. ConclusionsPentamethylquercetin inhibits angiotensin II-induced abdominal aortic aneurysm formation by bounding to C/EBPβ at Lys253. Therefore, PMQ prevents the formation of AAA and reduces the incidence of AAA.

Trimethylamine N-oxide promotes abdominal aortic aneurysm by inducing vascular inflammation and vascular smooth muscle cell phenotypic switching

ObjectiveInflammation and vascular smooth muscle cell (VSMC) phenotypic switching are implicated in the pathogenesis of abdominal aortic aneurysm (AAA). Trimethylamine N-oxide (TMAO) has emerged as a crucial risk factor in cardiovascular diseases, inducing vascular inflammation and calcification. We aimed to evaluate the effect of TMAO on the formation of AAA. Approach and resultsHere, we showed that TMAO was elevated in plasma from AAA patients compared with nonaneurysmal subjects by liquid chromatography‒mass spectrometry (LC‒MS) detection. Functional studies revealed that increased TMAO induced by feeding a choline-supplemented diet promoted Ang II-induced AAA formation. Immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), and Western blot analyses revealed that TMAO induced macrophage infiltration and inflammatory factor release. Conversely, inhibition of TMAO by supplementation with DMB suppressed AAA formation and the inflammatory response. Molecular studies revealed that TMAO regulated VSMC phenotypic switching. Flow cytometry analyses showed that TMAO induces macrophage M1-type polarization. Furthermore, pharmacological intervention experiments suggested that the nuclear factor-κB (NF-κB) signaling pathway was critical for TMAO to trigger AAA formation. ConclusionsTMAO promotes AAA formation by inducing vascular inflammation and VSMC phenotypic switching through activation of the NF-κB signaling pathway. Thus, TMAO is a prospective therapeutic AAA target.

CD147 monoclonal antibody attenuates abdominal aortic aneurysm formation in angiotensin II-Infused apoE-/- mice.

Abdominal aortic aneurysm (AAA) is a life threatening vascular disease. Our previous study reported the upregulation of CD147 expression in human aortic aneurysms. In this study, we injected apoE-/- mice intraperitoneally with CD147 monoclonal antibody or IgG control antibody to observe its effect on Angiotensin II (AngII) induced AAA formation. ApoE-/- mice were randomly divided into an AngⅡ+CD147 antibody group (n=20) and an AngⅡ+IgG antibody group (n=20). The Alzet osmotic minipump was implanted subcutaneously into the backs of mice to infuse AngII (1000ng/kg/min) for 28days and subsequently treated with CD147 monoclonal antibody or control IgG mAb (10μg/mouse/day) beginning one day after surgery. Body weight, food intake, drinking volume and blood pressure were measured weekly throughout the study. After 4weeks of injection, routine bloodwork measuring liver function, kidney function and lipid levels were recorded. Hematoxylin and eosin (H&E), Masson's trichrome, and Elastic van Gieson (EVG) staining were used to evaluate the pathological changes in blood vessels. In addition, Immunohistochemical assay was used to detect infiltration of inflammatory cells. Tandem mass tag (TMT)-based proteomic analysis was used to define differentially expressed proteins (DEPs) using a p-value<0.05 and fold change>1.2 or<0.83 as the threshold. Subsequently, we conducted protein-protein interaction (PPI) network and GO enrichment analysis to determine the core biological function altered after CD147 antibody injection. The CD147 monoclonal antibody suppresses Ang II-induced AAA formation in apoE-/- mice and reduced aortic expansion, elastic lamina degradation, and inflammatory cells accumulation. Bioinformatics analysis showed that Ptk6, Itch, Casp3, and Oas1a were the hub DEPs. These DEPs in the two group were mainly involved in collagen fibril organization, extracellular matrix organization, and muscle contraction. These data robustly demonstrated that CD147 monoclonal antibody suppresses Ang II-induced AAA formation through reduction of inflammatory response and regulation of the above defined hub proteins and biological processes. Thus, the CD147 monoclonal antibody might be a promising target in the treatment of abdominal aortic aneurysm.

ANGPTL8 Deletion Attenuates Abdominal Aortic Aneurysm Formation in ApoE-/- Mice.

Angiopoietin-like protein 8 (ANGPTL8) plays important roles in lipid metabolism, glucose metabolism, inflammation, and cell proliferation and migration. Clinical studies have indicated that circulating ANGPTL8 levels are increased in patients with thoracic aortic dissection (TAD). TAD shares several risk factors with abdominal aortic aneurysm (AAA). However, the role of ANGPTL8 in AAA pathogenesis has never been investigated. Here, we investigated the effect of ANGPTL8 knockout on abdominal aortic aneurysm (AAA) in ApoE-/- mice. ApoE-/-ANGPTL8-/- mice were generated by crossing ANGPTL8-/- and ApoE-/- mice. AAA was induced in ApoE-/- using perfusion of angiotensin II (AngII). ANGPTL8 was significantly upregulated in AAA tissues of human and experimental mice. Knockout of ANGPTL8 significantly reduced AngII-induced AAA formation, elastin breaks, aortic inflammatory cytokines, matrix metalloproteinase expression, and smooth muscle cell apoptosis in ApoE-/- mice. Similarly, ANGPTL8 sh-RNA significantly reduced AngII-induced AAA formation in ApoE-/- mice. ANGPTL8 deficiency inhibited AAA formation, and ANGPTL8 may therefore be a potential therapeutic target for AAA.

Celastrol Supplementation Ablates Sexual Dimorphism of Abdominal Aortic Aneurysm Formation in Mice.

Abdominal aortic aneurysms (AAAs) are permanent dilations of the abdominal aorta with 4-5 times greater prevalence in males than in females. The aim of this study is to define whether Celastrol, a pentacyclic triterpene from the root extracts of Tripterygium wilfordii, supplementation influences angiotensin II (AngII)-induced AAAs in hypercholesterolemic mice. Age-matched (8-12 weeks old) male and female low-density lipoprotein (Ldl) receptor-deficient mice were fed a fat-enriched diet supplemented with or without Celastrol (10 mg/kg/day) for five weeks. After one week of diet feeding, mice were infused with either saline (n = 5 per group) or AngII (500 or 1000 ng/kg/min, n = 12-15 per group) for 28 days. Dietary supplementation of Celastrol profoundly increased AngII-induced abdominal aortic luminal dilation and external aortic width in male mice as measured by ultrasonography and ex vivo measurement, with a significant increase in incidence compared to the control group. Celastrol supplementation in female mice resulted in significantly increased AngII-induced AAA formation and incidence. In addition, Celastrol supplementation significantly increased AngII-induced aortic medial elastin degradation accompanied by significant aortic MMP9 activation in both male and female mice compared to saline and AngII controls. Celastrol supplementation to Ldl receptor-deficient mice ablates sexual dimorphism and promotes AngII-induced AAA formation, which is associated with increased MMP9 activation and aortic medial destruction.

CCN2 deficiency in smooth muscle cells triggers cell reprogramming and aggravates aneurysm development.

Vascular smooth muscle cell (SMC) phenotypic switching is widely recognized as a key mechanism responsible for the pathogenesis of several aortic diseases, such as aortic aneurysm. Cellular communication network factor 2 (CCN2), often upregulated in human pathologies and animal disease models, exerts myriad context-dependent biological functions. However, current understanding of the role of SMC-CCN2 in SMC phenotypic switching and its function in the pathology of abdominal aortic aneurysm (AAA) is lacking. Here, we show that SMC-restricted CCN2 deficiency causes AAA in the infrarenal aorta of angiotensin II-infused (Ang II-infused) hypercholesterolemic mice at a similar anatomic location to human AAA. Notably, the resistance of naive C57BL/6 WT mice to Ang II-induced AAA formation is lost upon silencing of CCN2 in SMC. Furthermore, the pro-AAA phenotype of SMC-CCN2-KO mice is recapitulated in a different model that involves the application of elastase-β-aminopropionitrile. Mechanistically, our findings reveal that CCN2 intersects with TGF-β signaling and regulates SMC marker expression. Deficiency of CCN2 triggers SMC reprograming associated with alterations in Krüppel-like factor 4 and contractile marker expression, and this reprograming likely contributes to the development of AAA in mice. These results identify SMC-CCN2 as potentially a novel regulator of SMC phenotypic switching and AA biology.

Gasdermin D Deficiency in Vascular Smooth Muscle Cells Ameliorates Abdominal Aortic Aneurysm Through Reducing Putrescine Synthesis.

Abdominal aortic aneurysm (AAA) is a common vascular disease associated with significant phenotypic alterations in vascular smooth muscle cells (VSMCs). Gasdermin D (GSDMD) is a pore-forming effector of pyroptosis. In this study, the role of VSMC-specific GSDMD in the phenotypic alteration of VSMCs and AAA formation is determined. Single-cell transcriptome analyses reveal Gsdmd upregulation in aortic VSMCs in angiotensin (Ang) II-induced AAA. VSMC-specific Gsdmd deletion ameliorates Ang II-induced AAA in apolipoprotein E (ApoE)-/- mice. Using untargeted metabolomic analysis, it is found that putrescine is significantly reduced in the plasma and aortic tissues of VSMC-specific GSDMD deficient mice. High putrescine levels trigger a pro-inflammatory phenotype in VSMCs and increase susceptibility to Ang II-induced AAA formation in mice. In a population-based study, a high level of putrescine in plasma is associated with the risk of AAA (p < 2.2 × 10-16 ), consistent with the animal data. Mechanistically, GSDMD enhances endoplasmic reticulum stress-C/EBP homologous protein (CHOP) signaling, which in turn promotes the expression of ornithine decarboxylase 1 (ODC1), the enzyme responsible for increased putrescine levels. Treatment with the ODC1 inhibitor, difluoromethylornithine, reduces AAA formation in Ang II-infused ApoE-/- mice. The findings suggest that putrescine is a potential biomarker and target for AAA treatment.

Expression of a PCSK9 Gain-of-Function Mutation in C57BL/6J Mice to Facilitate Angiotensin II-Induced AAAs.

Angiotensin II (AngII) infusion in mice has been used widely to investigate mechanisms of abdominal aortic aneurysms (AAAs). To achieve a high incidence of AngII-induced AAAs, mice should be hypercholesterolemic. Therefore, either low-density lipoprotein receptor (LDLR) or apolipoprotein E deficiency have been used as a hypercholesterolemic background. However, it is a time-consuming and expensive process to generate compound deficient strains that have either an LDLR or apolipoprotein E deficient background. Proprotein convertase subtilisin/kexin type 9 (PCSK9) facilitates the degradation of LDL receptors. Previous studies demonstrated profound increases of plasma cholesterol concentrations after a single intraperitoneal injection of adeno-associated viruses (AAV) expressing a gain-of-function mutation of mouse PCSK9 (AAV.mPCSK9D377Y) in C57BL/6J mice fed a Western diet. Of note, injection of AAV.mPCSK9D377Y augmented AngII-induced AAA formation in C57BL/6J mice that had comparable severity of AAAs to LDLR deficient mice. Thus, AAV.mPCSK9D377Y infection greatly expedites studies on a gene of interest using AngII-induced AAAs. This commentary provides a brief technical guide of this approach and discusses the pros and cons of its use in AAA research.

Abstract 9079: Omentin Reduces Angiotensin II-Induced Abdominal Aortic Aneurysm Formation in Apolipoprotein E Knockout Mice

Background: Abdominal aortic aneurysm (AAA) is a life-threatening disease worldwide. Obesity is associated with an increased risk of AAA. Omentin is an adipokine, which is downregulated by obesity. Here we investigated the effect of omentin on angiotensin (Ang) II-induced AAA formation in apolipoprotein-E knockout (apoE-KO) mice. Methods and Results: ApoE-KO mice were crossed with transgenic mice expressing the human omentin in fat tissue (OMT-Tg mice). ApoE-KO/OMT-Tg and apoE-KO mice were subjected to continuous Ang II infusion. ApoE-KO/OMT-Tg mice exhibited a reduced maximal diameter of AAA compared with apo-E KO mice evaluated by macroscopic and ultrasound analyses. ApoE-KO/OMT-Tg mice showed a reduction of elastic fiber degradation in aorta compared with apoE-KO mice. ApoE-KO/OMT-Tg mice also displayed reduced expression levels of matrix metalloproteinase (MMP) 9, MMP2, pro-inflammatory mediators and macrophage surface marker, F4/80, in aortic walls compared with apo-E-KO mice. Conversely, intravenous administration of adenoviral vectors expressing omentin (Ad-OMT) significantly reduced maximal diameter and elastic fiber degradation of abdominal aorta after Ang II infusion in apoE-KO mice compared with control Ad-β-gal treatment. Treatment of human monocyte-derived macrophages with omentin protein attenuated activity and expression of MMP9 and NF-κB phosphorylation after stimulation with lipopolysaccharide. Treatment of human aortic smooth muscle cells with omentin protein attenuated activity and expression of MMP2 after stimulation with tumor necrosis factor α. The suppressive effects of omentin on MMP9 and MMP2 expression in macrophages and vascular smooth muscle cells were reversed by inhibition of PI3-kinase/Akt signaling or blockade of integrin αVβ3. Finally, continuous administration of PI3-kinase inhibitor LY294002 reversed the suppressive effects of Ad-OMT on maximal diameter and elastic fiber degradation of abdominal aorta in apoE-KO mice after Ang II infusion. Conclusion: Our data indicate that omentin acts as an adipokine that can attenuate Ang II-induced development of AAA by reducing MMP9 and MMP2 expression through the integrin αVβ3/PI3-kinase/Akt signaling in the vascular wall.

Abstract P107: Celastrol Supplementation Profoundly Activates Aortic Mmp-9 And Abolishes Sexual Dimorphism Of Abdominal Aortic Aneurysm In Mice.

Background and Objective: Abdominal Aortic Aneurysms (AAAs) are permanent dilations of the abdominal aorta with greater than 80% mortality after rupture. AAA prevalence is 4-5 times greater in males than females. AAA formation involves a complex process of destruction of aortic media through degradation of extracellular matrix proteins, elastin and collagen. Angiotensin II (AngII) infusion model of AAA in mice recapitulates major features of human AAA mainly male gender specificity. Celastrol, a pentacyclic triterpene from the root extracts of Thunder God Vine ( Tripterygium wilfordii ), strongly suppressed AngII-induced cardiovascular complications in mice. The purpose of this study is to test the effect of Celastrol supplementation on AngII-induced AAAs in mice. Methods and Results: Male and female LDL receptor -/- mice (8 weeks old; n= 12 per group) were fed a fat-enriched diet (21% wt/wt fat; 0.15% wt/wt cholesterol) supplemented with or without Celastrol (10mg/kg/day) for 5 weeks. After 1 week of diet feeding, mice were infused with AngII (500 or 1000 ng/kg/min) for 28 days by osmotic minipumps. Dietary supplementation of celastrol significantly promoted AngII-induced abdominal aortic luminal dilation (Con = 1.25 ± 0.05 versus Celas = 1.55 ± 0.09 mm, P&lt;0.05) and external aortic width (Con= 1.01 ± 0.09 versus Celas = 1.62 ± 0.14 mm, P&lt;0.05) in male mice as measured by ultra-sonography and ex vivo measurement, with 90% incidence (10/11) compared to 36% (4/11) in control group. Interestingly, celastrol supplementation to AAA female mice, resulted in a significant increase in AngII-induced aortic luminal dilation (Con = 1.17 ± 0.30 versus Celas = 1.57 ± 0.21 mm, P&lt;0.001) and AAA formation (Con= 0.94 ± 0.03 versus Celas = 1.36 ± 0.25 mm, P&lt;0.05) mice with 80% incidence (12/15) compared to 6% (1/15) in control group. Celastrol supplementation dramatically increased AngII-induced aortic leukocyte accumulation, MMP-9 activity and medial elastin degradation in both male and female mice compared to saline and AngII controls. Conclusion: These findings demonstrate that celastrol supplementation to LDLr -/- mice ablates sexual dimorphism and promotes AngII-induced AAA formation, which is associated with increased leukocytic infiltration and MMP-9 activation.

Silencing IL12p35 Promotes Angiotensin II-Mediated Abdominal Aortic Aneurysm through Activating the STAT4 Pathway.

Background and Purpose. Abdominal aortic aneurysm (AAA) is a chronic inflammatory disorder and the important causes of death among men over the age of 65 years. Interleukin-12p35 (IL12p35) is an inflammatory cytokine that participates in a variety of inflammatory diseases. However, the role of IL12p35 in the formation and development of AAA is still unknown. Experimental Approach. Male apolipoprotein E-deficient (Apoe−/−) mice were generated and infused with 1.44 mg/kg angiotensin II (Ang II) per day. We found that IL12p35 expression was noticeably increased in the murine AAA aorta and isolated aortic smooth muscle cells (SMCs) after Ang II stimulation. IL12p35 silencing promoted Ang II-induced AAA formation and rupture in Apoe−/− mice. IL12p35 silencing markedly increased the expression of inflammatory cytokines, including IL-1β, IL-6, and tumor necrosis factor-α (TNF-α), in both the serum and AAA aorta. Additionally, IL12p35 silencing exacerbated SMC apoptosis in Apoe−/− mice after Ang II infusion. IL12p35 silencing significantly increased signal transducer and activator of transcription (STAT) 4 phosphorylation levels in AAA mice, and STAT4 knockdown abolished the IL12p35-mediated proinflammatory response and SMC apoptosis. Interpretation. Silencing IL12p35 promotes AAA formation by activating the STAT4 pathway, and IL12p35 may serve as a novel and promising therapeutic target for AAA treatment.

MKL1 cooperates with p38MAPK to promote vascular senescence, inflammation, and abdominal aortic aneurysm

Abdominal aortic aneurysm (AAA) is a catastrophic disease with little effective therapy. Myocardin related transcription factor A (MRTFA, MKL1) is a multifaceted transcription factor, regulating diverse biological processes. However, a detailed understanding of the mechanistic role of MKL1 in AAA has yet to be elucidated. In this study, we showed induced MKL1 expression in thoracic and abdominal aneurysmal tissues, respectively in both mice and humans. MKL1 global knockout mice displayed reduced AAA formation and aortic rupture compared with wild-type mice. Both gene deletion and pharmacological inhibition of MKL1 markedly protected mice from aortic dissection, an early event in Angiotensin II (Ang II)-induced AAA formation. Loss of MKL1 was accompanied by reduced senescence/proinflammation in the vessel wall and cultured vascular smooth muscle cells (VSMCs). Mechanistically, a deficiency in MKL1 abolished AAA-induced p38 mitogen activated protein kinase (p38MAPK) activity. Similar to MKL1, loss of MAPK14 (p38α), the dominant isoform of p38MAPK family in VSMCs suppressed Ang II-induced AAA formation, vascular inflammation, and senescence marker expression. These results reveal a molecular pathway of AAA formation involving MKL1/p38MAPK stimulation and a VSMC senescent/proinflammatory phenotype. These data support targeting MKL1/p38MAPK pathway as a potential effective treatment for AAA.

Itaconate prevents abdominal aortic aneurysm formation through inhibiting inflammation via activation of Nrf2.

AbsractBackgroundIdentifying 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.MethodsQuantitative 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 was performed to detect the critical role of Nrf2 in the beneficial effect of OI on AAA.FindingsWe 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 effect. 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 was inhibited by itaconate.InterpretationItaconate inhibited AAA formation by suppressing vascular inflammation, and therapeutic approaches to increase itaconate are potentially beneficial for preventing AAA formation.FundingNational Natural Science Foundations of China and Guangzhou regenerative medicine and Health Laboratory of Guangdong.

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.

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.