Ang II-induced Abdominal Aortic Aneurysm Research Articles

Abstract 4137500: The Histone Methyltransferase MLL1 Regulates Notch Signaling and T Cell Phenotype During Pathologic Abdominal Aortic Aneurysm Development

Objective: Abdominal aortic aneurysms (AAA) are characterized by pathological vascular remodeling and an imbalance between anti-inflammatory regulatory T H cells (Tregs) and pro-inflammatory T H 17 cells within the aortic wall. The mechanisms regulating CD4 + T H cell differentiation during AAA development remain unknown. Recently, the histone methyltransferase, mixed lineage leukemia 1 (MLL1), has been shown to influence T H cell phenotype by directly altering key transcription factors for T H 17 (RORγ) and Treg (FOXp3) differentiation in varying disease states. As such the objective of this study was to evaluate the role of MLL1 in promoting T H 17 activity within AAA development and to determine key T H 17 cell signaling pathways that are altered Methods: Single-cell sequencing was conducted on human AAAs and control tissue samples. For our murine model, C57BL/6 mice were injected with an AAV encoding a PCSK9 gain-of-function mutation and fed a saturated fat diet followed by either AngII infusion to induce AAAs (1 µg/kg/min) or saline. Mice with T-cell specific deletion of Notch1 signaling (Notch1 f/f CD4 cre+ ) or MLL1 ( Mll1 f/f CD4 cre+ ) were subjected to the AngII-induced AAA model and AAA diameters quantified. CD4+ T-cells were isolated by MACs and gene expression analyzed by qPCR as well as T-cell phenotype by flow cytometry. ChIP was used to evaluate histone 3 lysine trimethylation (H3K4me3). Results: Single-cell RNA sequencing of human aortic tissue revealed that the epigenetic enzyme MLL1 and Notch1 signaling were profoundly upregulated in human AAA CD4+ cells compared to non-aneurysmal control samples. Congruently, CD4 + T H cells isolated from the murine AngII-induced AAA model displayed a predominance of T H 17 phenotype and elevated expression of MLL1. Histone methylation was evaluated by ChIP in AngII-induced AAA CD4+ cells and demonstrated increased levels of the MLL1-mediated activation histone methylation mark, H3K4me3, on the Notch 1 promoter. Mice deficient in CD4 + T H cell Notch signaling ( Notch1 f/f CD4 cre+ ) or MLL1 ( Mll1 f/f CD4 cre+ ) subjected to the AngII-induced murine model displayed significant reduction AAA development (n=10/group, p<0.05) with decreased CD4+ T H 17 cell activity and reciprocal increase in Treg expression. Conclusions: MLL1, a histone methyltransferase, regulates Notch1 signaling and T-cell differentiation in AAA development, causing pathologic inflammation. This pathway may serve as a potential therapeutic target to prevent aortic dilation.

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.

Divergent Effects of IL-18- and IL-18R-deficiency on Angiotensin II-induced Abdominal Aortic Aneurysm Formation

Currently, there are no pharmacological treatments for abdominal aortic aneurysms (AAA), and AAA rupture is fatal in ~90% of patients. Aortic inflammation is strongly correlated with the expansion of AAA and recently the NLRP3 inflammasome has been implicated in thedevelopment of AAA. Interleukin (IL)-18, an inflammasome-derived cytokine, exerts pro-inflammatory responses when bound to its cognate IL-18 receptor (IL-18Rα). The current project aimed to determine the effect of IL-18- and IL-18Rα-deficiency on the incidence of angiotensin (Ang) II-induced AAA. Ten-week-old male C57BL/6 (WT), IL-18-deficient ( Il18−/−) and IL-18Rα-deficient ( Il18r1−/−) mice were infused with Ang II (1.44 mg/kg/day, s.c.) for 28 days via osmotic minipump. Ang II-induced AAA incidence differed in all strains. While 52% of WT mice developed AAA (including premature deaths due to rupture), no AAAs were detected in Il18−/− mice (p<0.05, n=20-23). Strikingly, Il18r1−/− mice exhibited a higher incidence of AAA and associated mortality compared to WT mice(88%; p<0.05, n=23-25). Consistent with this finding, in vivo ultrasound imaging confirmed advanced dilation of the abdominal aorta at d7 after Ang II-treatment in Il18r1−/− mice cf. WT mice (1.49 ± 0.13 vs 1.15 ± 0.05 mm; p<0.05; n=23-25). Single cell RNA-sequencing of Ang II-infused mouse aorta revealed Il18r1 gene expression was limited to gamma-delta T cells, suggesting these cells may be driving IL-18 mediated AAA formation. In summary, IL-18-deficiency affords protection from AAA formation, while AAA incidence and severity were augmented in Il18r1−/− mice. Neutralisation of IL-18, but not IL-18Rα inhibition, may represent a novel therapeutic strategy to treat AAA. NHMRC (GNT1143674). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 3004: Intermittent Hypoxia Decreases Aortic Stiffness And Facilitates Abdominal Aortic Aneurysm

Introduction: Intermittent hypoxia (IH) is a phenomenon observed with obstructive sleep apnea (OSA), which is highly prevalent in patients with abdominal aortic aneurysm (AAA). However, it remains unknown if IH is a risk factor for the development of AAA. The goal of this study was to determine the effects of IH exposure on arterial stiffness, and to assess whether IH increases the susceptibility to angiotensin (Ang) II-induced AAA in mice. Methods: Male C57BL/6 mice (8-week-old) were subjected to IH (6.5%-21% O 2 , 90 s/cycle, 12 h/day) or room air (RA, 21% O 2 , 24 h/day) for short-term (6 weeks) or long-term (6 months) (n = 10/group). Short-term mice were examined for arterial stiffness by pulse wave velocity (PWV) and atomic force microscopy, while long-term mice were used for AAA studies. To induce AAA, mice were infused with Ang II (1 μg/kg/min) using osmotic minipumps during final 14 days of IH or RA exposure. Sham surgery was performed in control mice. Outcomes were assessed via transabdominal ultrasound imaging and histology. Results: Short-term IH exposure decreased the stiffness of the abdominal (0.88 vs. 2.32 m/s in RA; p=0.0006 ) and thoracic (2.0 vs. 3.5 kPa in RA; p=0.01 ) aorta. Ang II increased abdominal aortic diameter in RA mice compared to sham and long-term IH with Ang II further augmented the aortic diameter (1.56 vs. 1.34 mm in RA Ang II; p=0.03 ) and incidence of AAA (50 vs. 10% in RA Ang II; p=0.05 ). Histologically, long-term IH plus Ang II augmented medial elastin fragmentation (7.7 vs. 5.3 in RA Ang II; p=0.04 ) and aortic collagen content (3.1 vs. 1.3 in RA Ang II; p=0.06 ). Ang II tended to non-significantly increase PWV in both groups (RA sham 1.9 vs. RA Ang II 2.5 and IH Ang II 2.3 m/s). Notably, a negative correlation between maximum aortic diameter and PWV was observed in AAA-induced mice (R 2 =0.48, p=0.0009 ). Conclusions: IH favors AAA development in association with changes in aortic stiffness. These findings support a potential causal link between OSA and AAA pathogenesis. Clarification of the molecular mechanisms may reveal novel therapeutic targets for the management of AAA.

Abstract 149: The Protective Effect Of Vamp8 Deficiency In Aortopathies: Potential Impact Of Impaired Platelet Cargo Release

Introduction and Objective: Platelet activation and subsequent cargo secretion play a critical role in thrombus formation and aortic remodeling, factors potentially influencing the growth and rupture of abdominal aortic aneurysms (AAA). Despite the compelling data that platelet activation is associated with AAA and rupture, the underlying mechanisms remain poorly understood. This study hypothesizes that VAMP8, a key element of platelets’ secretory machinery, significantly contributes to AAA formation. Approach and Results: A retrospective, single-center multiple comparisons study of adult aneurysmal patients admitted to UKHealthCare between 2004 and 2023 revealed a significantly lower platelet count in AAA patients (n=3131, 213.76 ± 3.46) vs. healthy individuals (n= 6309, 243.18 ± 4.09) suggesting an increase in platelets consumption, potentially in the aneurysmal sack. Thoracic aortic aneurysm and aortic dissection patients also indicated lower platelet count. In vivo , platelets were shown to accumulate at elastin break sites and false lumens of abdominal aortas from 28-day AngII-infused hypercholesteremic mice. To assess the role of platelets, specifically platelet secretion, we utilized VAMP8 null mice. We first validated the platelet secretion defect in these mice and then used RNASeq and proteomics array to define the role of VAMP8 in platelet cargo packaging. Next, VAMP8 -/- or WT hypercholesterolemic male mice fed a Western diet were infused with AngII (1,000 ng/kg/min) for 4 weeks. Our results revealed that VAMP8 -/- mice are protected against AngII-driven aortic rupture. Additionally, aortic ex vivo analysis indicated that the VAMP8 deficiency (n=15, aortic diameter: 0.9 mm ± 0.03) profoundly attenuated AngII-induced AAA compared to control (n=9, aortic diameter: 2.0 mm ± 0.2). Further analysis also indicated a marked reduction in thoracic aortic aneurysm and atherosclerosis development in VAMP8 -\- vs. Control group. Conclusion: Our results elucidate that a deficiency in VAMP8 results in the profound attenuation of aortic aneurysms and atherosclerosis development, introducing a novel paradigm for understanding the impact of platelet cargo secretion in the development of aortopathies.

Abstract 1001: Interleukin-33 Deficiency Exacerbated Angiotensin II-induced Abdominal Aortic Aneurysms

Objective: Chronic infusion of angiotensin II (AngII) promotes development of abdominal aortic aneurysms (AAAs) in Apolipoprotein E-deficient (ApoE-/-) mice. Interleukin 33 (IL-33), known as one of IL-1 family, is rapidly released from damaged endothelial and epithelial cells. IL-33 binds to its receptor, ST2, and exerts various physiological functions through its downstream signaling. Previous studies have shown that exogenous IL-33 attenuates development of AAAs through wound healing resolution. The purpose of this study was to evaluate whether IL-33 deficiency exacerbated development of AAAs by using IL-33xApoE double deficient (IL-33-/-xApoE-/-) mice. Methods and Results: Male ApoE-/- and IL-33-/-xApoE-/- (8-12 weeks old) mice were assigned into saline infused group (ApoE-/- n=5, IL-33-/-xApoE-/- n=7) and AngII infused group (ApoE-/- n=19, IL-33-/-xApoE-/- n=19). Mice were infused subcutaneously with either saline or AngII by osmotic minipumps for 4 weeks. During 4-week AngII infusion, 3 mice died by AAA rupture in each AngII-infused group. There was no significant difference in ex vivo abdominal aortic width between saline infused groups (ApoE-/- 0.87±0.17 mm vs IL-33-/-xApoE-/- 0.87±0.14 mm, n.s.) nor body weight (ApoE-/- 27.3±0.9 g vs IL-33-/-xApoE-/- 27.9±0.9 g, n.s.) but serum total cholesterol concentrations in IL-33-/-xApoE-/- was higher than in ApoE-/- (522±85 mg/dL vs 427±108 mg/dL, p=0.026). AngII equally elevated systolic blood pressure (ApoE-/- 151±15 mmHg, IL-33-/-xApoE-/- 155±17 mmHg, n.s.). AngII increased ex vivo maximum abdominal aortic width in AngII infused groups, but AAAs were further exacerbated in the group of IL-33-/-xApoE-/- mice (ApoE-/- 1.31±0.09 mm vs IL-33-/-xApoE-/- 1.76±0.09 mm, p=0.001). In addition, the severity of AAAs in AngII infused groups was significantly worse in IL-33-/-xApoE-/- than in ApoE-/- (p=0.037). Conclusion: Our study demonstrated IL-33 deficiency exacerbated AngII-induced AAAs in male ApoE-/- mice.

Abstract 1005: Hypertriglyceridemia Promotes Aortic Aneurysm Formation And Rupture In Angiotensin Ii Infused Mice

Plasma total cholesterol (TC) and triglyceride (TG) are reported to be positively associated with the risk of abdominal aortic aneurysm (AAA). Our group discovered that hypercholesterolemia augments angiotensin II-induced AAA in mice. We also found that remnants of triglyceride-rich lipoproteins (TRLs) may be crucial for AngII-induced AAA. Recently, it was reported that hypertriglyceridemia (HTG) caused by inducible lipoprotein lipase deficiency (iLpl-/-) results in uptake and accumulation of TRL lipid in aortic endothelial cells in vivo. Based upon these findings, we hypothesized that HTG stimulates AAA formation. Methods: Adult male and female Lplf/f.beta-actin-Mer/Cre/Mer 1/0 and Lplf/f mice were administered 75 mg/kg/day tamoxifen for 5 consecutive days. Mice were fed either a standard diet (SD) throughout the study or a Western-type diet (WD) starting 1 wk after the completion of tamoxifen administration and continuing for a total of 5 wks. Mini osmotic pumps were implanted in mice 2 wks after completion of tamoxifen administration and delivered saline or AngII at 1,000 ng/kg/min for 4 wks. Results: AngII infused and SD fed iLpl-/- versus Lplf/f mice had elevated plasma TG and TC levels but similar abdominal aortic external diameter and AAA incidence. Since the SD was low in fat, a high fat WD was then fed to AngII-infused mice. Plasma TG and TC were increased in female iLpl-/- versus Lplf/f mice. However, plasma lipid concentrations could not be measured in male iLpl-/- because 10 of 11 animals had died from aortic rupture. In contrast, none of the male Lplf/f mice died and only 4 of 11 had abdominal aortic dilation. Female compared to male iLpl-/- mice were protected from AngII-induced aortic rupture (1/11 died) and only 1 of 9 females had abdominal aortic dilation. To eliminate the possibility that HTG alone caused aortic rupture in male mice, the study was repeated with the addition of saline infusion. In agreement with the first study, AngII plus WD lead to death by aortic rupture in all male iLpl-/- mice (9/9). In contrast, iLpl-/- male mice infused with saline had markedly greater survival (1/5 died of unknown cause). Conclusions: HTG causes aortic aneurysm development in AngII-infused male iLpl-/- mice. Thus, treating HTG could reduce AAA risk.

Abstract 3007: The Role Of Myeloperoxidase In The Development Of Abdominal Aortic Aneurysm

Background: Abdominal aortic aneurysm (AAA) is a life-threatening cardiovascular disorder characterized by inflammation in the aortic media, extracellular matrix degradation, aortic dilation and rupture. Increasing evidence identifies a role for oxidative stress, although the oxidative reactions involved requires clarification. Myeloperoxidase (MPO) is a pro-inflammatory neutrophil-derived enzyme that promotes cardiovascular disease by catalyzing tissue damaging oxidative reactions. Here we investigated the impact of MPO pharmacological inhibition and gene deletion on AAA. For pharmacological inhibition, the clinically trialed 2-thioxanthine class of irreversible MPO inhibitors was employed. Methods: Apolipoprotein E (ApoE -/- ) or ApoE -/- /MPO -/- gene-knockout mice were infused with angiotensin II (AngII) by osmotic mini-pumps for 28 days and received 2-thioxanthine or vehicle via daily oral gavage. AAA development was monitored by micro-ultrasound measurement of aortic diameter and post-mortem measurement of maximal aortic width at the supra-renal aorta. Atherosclerosis was measured at the aortic sinus by morphometry and aortic arch by en face analysis of oil-red-O-stained lipids. Cardiac fibrosis was measured by picrosirius red staining. Results: AngII infusion afforded an AAA incidence rate of 60% in ApoE -/- mice that was reduced to 25% by 2-thioxanthine treatment. 2-Thioxanthines also inhibited the AngII-mediated increase in aortic diameter and cardiac fibrosis, but not the AngII-induced accelerated development of atherosclerosis at the aortic sinus and arch. Paradoxically, studies in ApoE -/- /MPO -/- mice established that MPO gene-deletion did not affect AngII-induced AAA but significantly attenuated the increase in aortic arch atherosclerosis. Notably, 2-thioxanthines significantly inhibited AAA incidence and severity in AngII-infused ApoE -/- /MPO -/- mice. Conclusion: These studies highlight the complexity surrounding the role of MPO in a murine model of AAA and atherosclerosis. Importantly, this work establishes that 2-thioxanthines can protect against AAA in the absence of MPO, supporting that this clinically trialed MPO inhibitor drug class can target other heme peroxidases in vivo .

Spatiotemporal ATF3 Expression Determines VSMC Fate in Abdominal Aortic Aneurysm.

Abdominal aortic aneurysm (AAA) is a catastrophic disease with little effective therapy, likely due to the limited understanding of the mechanisms underlying AAA development and progression. ATF3 (activating transcription factor 3) has been increasingly recognized as a key regulator of cardiovascular diseases. However, the role of ATF3 in AAA development and progression remains elusive. Genome-wide RNA sequencing analysis was performed on the aorta isolated from saline or Ang II (angiotensin II)-induced AAA mice, and ATF3 was identified as the potential key gene for AAA development. To examine the role of ATF3 in AAA development, vascular smooth muscle cell-specific ATF3 knockdown or overexpressed mice by recombinant adeno-associated virus serotype 9 vectors carrying ATF3, or shRNA-ATF3 with SM22α (smooth muscle protein 22-α) promoter were used in Ang II-induced AAA mice. In human and murine vascular smooth muscle cells, gain or loss of function experiments were performed to investigate the role of ATF3 in vascular smooth muscle cell proliferation and apoptosis. In both Ang II-induced AAA mice and patients with AAA, the expression of ATF3 was reduced in aneurysm tissues but increased in aortic lesion tissues. The deficiency of ATF3 in vascular smooth muscle cell promoted AAA formation in Ang II-induced AAA mice. PDGFRB (platelet-derived growth factor receptor β) was identified as the target of ATF3, which mediated vascular smooth muscle cell proliferation in response to TNF-alpha (tumor necrosis factor-α) at the early stage of AAA. ATF3 suppressed the mitochondria-dependent apoptosis at the advanced stage by upregulating its direct target BCL2. Our chromatin immunoprecipitation results also demonstrated that the recruitment of NFκB1 and P300/BAF/H3K27ac complex to the ATF3 promoter induces ATF3 transcription via enhancer activation. NFKB1 inhibitor (andrographolide) inhibits the expression of ATF3 by blocking the recruiters NFKB1 and ATF3-enhancer to the ATF3-promoter region, ultimately leading to AAA development. Our results demonstrate a previously unrecognized role of ATF3 in AAA development and progression, and ATF3 may serve as a novel therapeutic and prognostic marker for AAA.

Smooth-Muscle-Cell-Specific Deletion of CD38 Protects Mice from AngII-Induced Abdominal Aortic Aneurysm through Inhibiting Vascular Remodeling.

Abdominal aortic aneurysm (AAA) is a serious vascular disease which is associated with vascular remodeling. CD38 is a main NAD+-consuming enzyme in mammals, and our previous results showed that CD38 plays the important roles in many cardiovascular diseases. However, the role of CD38 in AAA has not been explored. Here, we report that smooth-muscle-cell-specific deletion of CD38 (CD38SKO) significantly reduced the morbidity of AngII-induced AAA in CD38SKOApoe-/- mice, which was accompanied with a increases in the aortic diameter, medial thickness, collagen deposition, and elastin degradation of aortas. In addition, CD38SKO significantly suppressed the AngII-induced decreases in α-SMA, SM22α, and MYH11 expression; the increase in Vimentin expression in VSMCs; and the increase in VCAM-1 expression in smooth muscle cells and macrophage infiltration. Furthermore, we demonstrated that the role of CD38SKO in attenuating AAA was associated with the activation of sirtuin signaling pathways. Therefore, we concluded that CD38 plays a pivotal role in AngII-induced AAA through promoting vascular remodeling, suggesting that CD38 may serve as a potential therapeutic target for the prevention of AAA.

Antisense oligonucleotide targeting hepatic Serum Amyloid A limits the progression of angiotensin II-induced abdominal aortic aneurysm formation

Background and aimsObesity increases the risk for abdominal aortic aneurysms (AAA) in humans and enhances angiotensin II (AngII)-induced AAA formation in C57BL/6 mice. We reported that deficiency of Serum Amyloid A (SAA) significantly reduces AngII-induced inflammation and AAA in both hyperlipidemic apoE-deficient and obese C57BL/6 mice. The aim of this study is to investigate whether SAA plays a role in the progression of early AAA in obese C57BL/6 mice. MethodsMale C57BL/6J mice were fed a high-fat diet (60% kcal as fat) throughout the study. After 4 months of diet, the mice were infused with AngII until the end of the study. Mice with at least a 25% increase in the luminal diameter of the abdominal aorta after 4 weeks of AngII infusion were stratified into 2 groups. The first group received a control antisense oligonucleotide (Ctr ASO), and the second group received ASO that suppresses SAA (SAA-ASO) until the end of the study. ResultsPlasma SAA levels were significantly reduced by the SAA ASO treatment. While mice that received the control ASO had continued aortic dilation throughout the AngII infusion periods, the mice that received SAA-ASO had a significant reduction in the progression of aortic dilation, which was associated with significant reductions in matrix metalloprotease activities, decreased macrophage infiltration and decreased elastin breaks in the abdominal aortas. ConclusionsWe demonstrate for the first time that suppression of SAA protects obese C57BL/6 mice from the progression of AngII-induced AAA. Suppression of SAA may be a therapeutic approach to limit AAA progression.

Terazosin attenuates abdominal aortic aneurysm formation by downregulating Peg3 expression to inhibit vascular smooth muscle cell apoptosis and senescence

Abdominal aortic aneurysm (AAA), a vascular degenerative disease, is a potentially life-threatening condition characterised by the loss of vascular smooth muscle cells (VSMCs), degradation of extracellular matrix (ECM), inflammation, and oxidative stress. Despite the severity of AAA, effective drugs for treatment are scarce. At low doses, terazosin (TZ) exerts antiapoptotic and anti-inflammatory effects in several diseases, but its potential to protect against AAA remains unexplored. Herein, we investigated the effects of TZ in two AAA animal models: Angiotensin II (Ang II) infusion in Apoe−/− mice and calcium chloride application in C57BL/6J mice. Mice were orally administered with TZ (100 or 1000 μg/kg/day). The in vivo results indicated that low-dose TZ alleviated AAA formation in both models. Low-dose TZ significantly reduced aortic pulse wave velocity without exerting an apparent antihypertensive effect in the Ang II-induced AAA model. Paternally expressed gene 3 (Peg3) was identified via RNA sequencing as a novel TZ target. PEG3 expression was significantly elevated in both mouse and human AAA tissues. TZ suppressed PEG3 expression and reduced the abundance of matrix metalloproteinases (MMP2/MMP9) in the tunica media. Functional experiments and molecular analyses revealed that TZ (10 nM) treatment and Peg3 knockdown effectively prevented Ang II-induced VSMC senescence and apoptosis in vitro. Thus, Peg3, a novel target of TZ, mediates inflammation-induced VSMC apoptosis and senescence. Low-dose TZ downregulates Peg3 expression to attenuate AAA formation and ECM degradation, suggesting a promising therapeutic strategy for AAA.

A preliminary study of sirtuin-1 on angiotensin II-induced senescence and inflammation in abdominal aortic aneurysms.

Recent evidence suggests the involvement of senescence and inflammation in abdominal aortic aneurysm (AAA). Considering the role of sirtuin-1 (SIRT1) in delaying senescence, we aimed to preliminarily investigate the potential mechanism underlying the effects of SIRT1 in senescence and inflammation during AAA. A cell AAA model was established using angiotensin II (Ang II) as the inducer, which was applied to treat human aortic vascular smooth muscle cells (HASMCs). The senescence and cell cycle of treated HASMCs were evaluated based on senescence-associated (SA)-b-galactosidase (b-gal) assay and flow cytometry, respectively. The levels of inflammatory cytokines and proteins related to senescence-associated secretory phenotype (SASP), along with nuclear factor-kappa B (NF-kB) and mitogen-activated protein kinases (MAPK) pathways, as well as SIRT1, were gauged. The correlation between SIRT1 and NF-kB and MAPK pathway-related proteins was further estimated. In Ang II-treated HASMCs, reduced SIRT1 and B-cell lymphoma-2 levels yet increased levels of SASP-related proteins P16 and P21, inflammatory cytokines, as well as Bax and caspases were all visible. In the meantime, Ang II exposure enhanced the number of b-gal-positive HASMCs and promoted cell cycle arrest. SIRT1 was also repressed following Ang II treatment and negatively correlated with NF-kB and MAPK pathway-related proteins (P < 0.05). Furthermore, the overexpression of SIRT1 diminished the levels of SASP-related proteins and reduced the phosphorylation of extracellular regulated kinase 1/2 and P65 in Ang II-treated HASMCs (P < 0.05). Taken together, our results indicate that SIRT1 overexpression attenuates the inflammatory and senescent responses of HASMCs in the Ang II-induced AAA cell model. This finding suggests that SIRT1 can be a highly promising target for clinical treatment 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.

Neutrophil Elastase Inhibition by Sivelestat (ONO-5046) Attenuates AngII-Induced Abdominal Aortic Aneurysms in Apolipoprotein E-Deficient Mice.

Abdominal aortic aneurysm (AAA) is an arterial disease characterized by dilatation of the aortic wall. It has been suggested that neutrophil counts and neutrophil elastase activity are associated with AAA. We investigated whether a neutrophil elastase (NE) inhibitor, sivelestat (Siv), had a protective effect against angiotensin II (AngII)-induced AAAs. Male apolipoprotein E-deficient mice were assigned into three groups: Vehicle + saline, AngII + saline, and AngII + Siv. All mice were administered intraperitoneally with either Siv or vehicle twice daily after AngII infusion. In the 4-week AngII infusion study, plasma NE concentration (P = 0.041) and its activity (P = 0.011) were elevated by AngII. These increases were attenuated by Siv (concentration:P = 0.010, activity:P = 0.027). Further, plasma elastase activity was closely correlated with aortic width (R = 0.6976, P < 0.001). In the 1-week AngII infusion study, plasma and tissue elastase activity increased by AngII (plasma:P = 0.034, tissue:P < 0.001), but were reduced by Siv (plasma:P = 0.014, tissue:P = 0.024). AngII increased aortic width (P = 0.011) but was attenuated by co-administration of Siv (P = 0.022). Moreover, Siv decreased the incidence of AAAs (P = 0.009). Elastin fragmentation induced by AngII was reduced by Siv. Many inflammatory cells that were either CD68 or Gr-1 positive were observed in the AngII + saline group, whereas few inflammatory cells were accumulated in the AngII + Siv group. MMP-2 and MMP-9 were enhanced by AngII, but were reduced by Siv. In vitro, MMP-2 activity was induced by human NE (medium:P < 0.001, cells:P = 0.001), which was attenuated by co-incubation of Siv in medium (P < 0.001) and protein of human aortic smooth muscle cells (P = 0.001). Siv attenuated AngII-induced AAA through the inhibition of NE.

Melanocortin-4 receptor in macrophages attenuated angiotensin II-induced abdominal aortic aneurysm in mice

Obesity is recognized as an independent risk factor for abdominal aortic aneurysm (AAA). While mutations in the melanocortin-4 receptor (MC4R) gene is the most common cause of obesity caused by mutations in a single gene, the link between MC4R function and vascular disease has still remained unclear. Here, by using melanocortin-4 receptor (MC4R) deficient mice, we confirmed MC4R deficiency promotes AAA and atherosclerosis. We demonstrated the contribution of two novel factors towards vascular vulnerability in this model: leptin signaling in vascular smooth muscle cells (VSMCs) and loss of MC4R signaling in macrophages. Leptin was shown to promote vascular vulnerability via PI3K-dependent upregulation of Spp1 expression in VSMC. Additionally, Ang II-induced AAA incidence was significantly reduced when MC4R gene expression was myeloid cell-specifically rescued in MC4R deficient (MC4RTB/TB) mice. Ex vivo analysis showed a suppression in NF-κB activity in bone marrow-derived macrophages from LysM(+);MC4RTB/TB mice compared to LysM(−);MC4RTB/TB mice, which exaggerates with endogenous MC4R ligand treatment; α-MSH. These results suggest that MC4R signaling in macrophages attenuates AAA by inhibiting NF-κB activity and subsequent vascular inflammation.

Abstract 12885: Pathological Perk/Atf4 Activation in Abdominal Aortic Aneurysm Formation

Introduction: Abdominal aortic aneurysms (AAA) are characterized by vascular smooth muscle cell (VSMC) apoptosis leading to pathological aortic remodeling. Activation of the endoplasmic reticulum (ER) stress response has been linked to VSMC dysfunction but the driving factors behind ER stress activation and its role on AAA development remains poorly defined. The epigenetic enzyme, MLL1, plays a critical role in establishing cellular phenotype and regulating gene expression. We examine the MLL-mediated epigenetic regulation of the ER stress response in VSMCs during AAA development and establish the ER stress pathways as a novel target in the treatment AAAs. Methods: Single-cell sequencing was conducted on human AAA and control tissue samples. For our murine model, C57BL/6 mice were injected with an AAV encoding a PCSK9 gain-of-function mutation and then fed a saturated fat-enriched diet and infused with AngII (1 μg/kg/min) with or without 4-PBA (20mg/kg/day). The elastase induced AAA model was used as a secondary murine model. AAA maximum diameters were quantified and VSCMs were isolated. Eif2a, Atf4 , and Chop gene expression was examined. Apoptosis was quantified by Annexin V staining. Results: Using single-cell RNA sequencing of human aortic tissue, we identified EIF2A, ATF4 , and CHOP were profoundly upregulated in aneurysmal VSMCs as well as two murine AAA models. Mechanistically, TNFα signaling in VSMCs leads to activation of MLL1 which trimethylates histone 3 lysine 4 (H3K4me3) at NF-κB binding sites on the EIF2A and ATF4 gene promoters thereby upregulating the ER stress response and driving VSMC apoptosis. In vivo pharmacological inhibition of the eIF2A/ATF4 ER stress response via 4-PBA injection inhibited AAA formation in both the AngII-induced and elastase-induced AAA model resulting with in significant reduction in VSMC apoptosis, elastin fragmentation, and inflammatory cytokine infiltration. Conclusions: Taken together, our results identify MLL1-mediated activation of the eIF2A/ATF4 ER stress response as a critical regulator of VSMC apoptosis in AAA formation and suggest inhibition of ER stress response as a mechanistic target for the management of AAAs.