Abdominal Aortic Aneurysm Severity Research Articles

Insights on P2X7 in patients with abdominal aortic aneurysm and carotid atherosclerosis

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Ministero della Salute, Italia, Rete Cardiologica Nazionale Background Carotid atherosclerosis and abdominal aortic aneurysm (AAA) are co-morbidities [1]. P2X-purinoceptor-7 (P2X7) is expressed both in human AAA and atherosclerotic carotid plaque (CPL) and is instability-associated in CPL [2-4]. P2X7 is functionally related to miR-150 and miR-186 (two microRNA which modulate vascular cell proliferation) [5,6], and may be shed into the bloodstream in correlation with C-reactive protein increase [7]. Little is known about the possible association between AAA risk and circulating P2X7, miR-150 and miR-186 in patients with/without hemodinamically significant CPL. Purpose Our aim is exploring the possible relationship between circulating P2X7, target miRs clinical status and lesion dimensions in patients with CPL and/or AAA. Methods Male patients with AAA and/or CPL were characterized.CPL stenosis was assessed by Ecocolordoppler-TSA. Eligible cohort included 20 patients with AAA undergoing aneurysmectomy [10 with CPL stenosis ≤40% (group A), 10with CPL stenosis >40% (group B)], 10 patients with hemodinamically significant CPL and small or no AAA undergoing endarterectomy (group C) donating blood and vascular samples at surgery. Twelve healthy blood donors (group D) were enrolled as controls. P2X7 protein was evaluated by ELISA. P2X7 gene, miR-150 and miR-186 were determined by RT-qPCR. Results In group A, CPL appeared mainly localized at carotid bulb, in group B extended along branches. P2X7 gene was undetermined into serum and expressed without differences among groups in patient tissues. P2X7 protein was measurable in all serum samples and was lower in group B than in the others (p=0,0117, p=0,0009, p=0,0003 vs. group A, C, D, respectively) and in group C vs. D (p=0,0006). These data differentiated patients with hemodinamically significant CPLs with severe AAA from the other patients and all those with hemodinamically significant CPLs (irrespectively from AAA severity) from healthy individuals. In group B but not in the others, serum P2X7 was linearly associated to platelet concentration. In group C the amounts of P2X7 in CPL tissue and serum were negatively associated, whereas in group A and B the P2X7 levels in AAA tissue and serum were unrelated. The miRs were detected into both serum and tissue and not correlated among them nor with circulating P2X7 protein. The serum miR-186, but not miR-150, was differentially expressed in group A vs. B (p=0,0266) and linearly related to AAA diameter in group B (p=0.0351). P2X7 content and expressed gene (p=0,015, p= 0,023, respectively) and miR expressions (p=0,0418 for miR-186, p=0,0012 for miR-150) were more elevated in AAA vs. CPL tissues, highlighting the vascular heterogenicity. Conclusions Preliminary data suggest a role for P2X7 and miR-186 in profiling patients with hemodinamically significant CPL with/without high risk AAA, deserving further investigations.

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.

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.

Abstract 12379: AT1-AA (Angiotensin II Type 1 Receptor Autoantibody) Aggravates Abdominal Aortic Aneurysm Through Activating AT1R/Gq Allosterically

Background: Angiotensin II type 1 receptor (AT1R) is a key mediator of the rennin-angiotensin-aldosterone system (RAAS). Autoantibodies against AT1R (AT1-AA), as an activator of AT1R, was found in abdominal aortic aneurysm (AAA) related diseases, such as hypertension and atherosclerosis. However, the role of AT1-AA in AAA is still unclear. Methods: We used angiotensin II (AngII)-infused male ApoE -/- mice model of AAA. Ultrasonography measurement, histological assessment, western blot, qRT-PCR and RNA sequencing were used to assess the severity of AAA. Nanoluciferase-based bioluminescence resonance energy transfer (NanoBRET) assay, radio assay of receptors and homogeneous time resolved fluorescence (HTRF) were used to measure the activation feature of AT1R and the affinity of AT1R with AT1-AA or AngII. Results: AT1-AA increased the incidence of AAA (vehicle vs. AT1-AA: 47% vs. 81%; P<0.05), which can be attenuated by AT1R blocker Candesartan. Furthermore, AT1-AA greatly increased severity of AAA, due to augmenting the diameter of abdominal aorta and the degradation of elastin by enhancing the expression of MMP2/9 (matrix metalloproteinase) and the accumulation of inflammatory cells. And AT1-AA could induce phenotypic switch of smooth muscle cells. But AT1-AA does not increase the plasma level of AngII. Mechanically, AT1-AA enhanced AngII-induced recruitment of Gq, but not β-arrestin1/2. AT1-AA potentiated the AngII-mediated Gq-dependent inositol phosphate (EC50 shift=3.829 nM) and the activation of PKC and ERK1/2. The responses were abolished by AT1R blocker and Gq inhibitor. The affinity of AT1-AA for the AT1R in cells was calculated to be 75.49 ± 4.7 nM. AT1-AA cannot compete for the binding of AngII to AT1R. However, AT1-AA significantly potentiated the binding of AngII with AT1R allosterically (mean K D ± SEM: 34.11 ± 0.9765 nM at 0 nM AT1-AA vs. 25.63 ± 1.826 nM at 100 nM AT1-AA; p<0.05), without altering the maximum binding. Conclusion: AT1-AA, as an allosteric activator of AT1R, promotes AAA through increasing the affinity of AngII with AT1R and activating Gq biasedly.

Abstract 15171: Endothelium-Derived C-Type Natriuretic Peptide Prevents the Development and Progression of Abdominal Aortic Aneurysm

Background: Abdominal aortic aneurysm (AAA) is a life-threatening disease characterised by dilatation, inflammation and structural weakness of the abdominal aorta. Since the only effective existing intervention is surgery, development of pharmacological therapies is desperately needed. C-type natriuretic peptide (CNP) is a key endothelium-derived molecule that possesses vasodilator, anti-inflammatory and anti-atherogenic activity. Thus, we explored the role and therapeutic potential of CNP in AAA pathogenesis. Methods: Suprarenal aortic diameter was assessed by ultrasonography at baseline and following Angiotensin II (AngII, 1.44mg/kg/day) infusion in wild type (WT) or endothelium-restricted CNP deficient (ecCNP -/- ) mice infected with an AAV-expressing proprotein convertase subtilisin/kexin type 9 gain-of-function mutation (D377Y) or backcrossed on an apolipoprotein E deficient (ApoE -/- ) background. At 28 days, aortas were harvested for qRT-PCR, pharmacological assessment of vasorelaxant activity and histological analysis; plasma CNP concentrations were also determined. In some studies, CNP (0.2mg/kg/day) was infused to rescue any adverse phenotype. Results: ecCNP -/- exhibited an aggravated AAA phenotype compared to WT, exemplified by greater dilation, fibrosis and elastin degradation (without changes in blood pressure). Whilst plasma CNP concentrations were reduced in murine AAA, CNP mRNA abundance was elevated within aneurysmal tissue of ecCNP -/- , accompanied by increased abundance of mRNA encoding markers of inflammation ( IL-6, Adgre1, VCAM-1, CCL-2 ), extracellular matrix remodelling/calcification ( MMP-2, Notch-1, BMPR2, smoothelin ), fibrosis ( fibronectin ) and apoptosis ( Bax ) . CNP-induced relaxations were enhanced in aortic rings from aneurysmal tissue compared to control; an effect possibly underpinned by upregulation of natriuretic peptide receptor expression. Prophylactic administration of CNP protected against the severity of AngII-induced AAA. Conclusions: Endothelial CNP plays an important role in attenuating AAA formation by preserving aortic structure and function. Therapeutic strategies aimed at mimicking CNP bioactivity may prevent the clinical progression of AAA.

Pharmacological Inhibition of Gasdermin D Suppresses Angiotensin II-Induced Experimental Abdominal Aortic Aneurysms.

Gasdermin D, a molecule downstream of the nucleotide-binding oligomerization domain-like receptor family pyrin domain containing inflammasome, forms the membrane pore for the secretion of interleukin (IL)-1β and IL-18, and also mediates pyroptosis. This study was to explore the influence of treatment with disulfiram, a small molecule inhibitor to gasdermin D, on the formation and progression of experimental abdominal aortic aneurysms (AAA). AAAs were induced in 10-week-old male apolipoprotein E deficient mice by subcutaneous infusion of angiotensin II (1000 ng/min/kg body weight) for 28 days via osmotic minipumps. Three days prior to angiotensin II infusion, disulfiram (50 mg/kg) or an equal volume of saline as the vehicle control was administered daily via oral gavage. The influence on experimental AAAs was analyzed by serial measurements of aortic diameters via ultrasonography, grading AAA severity and histopathology at sacrifice. Serum IL-1β and IL-18 levels, systolic blood pressure, total cholesterol, and triglyceride were also measured. Additional experiments assayed the influences on the cell viability and IL-1β secretion of in vitro activated macrophages. Disulfiram significantly reduced the enlargement, incidence, and severity of angiotensin II-induced experimental AAAs with attenuation of medial elastin breaks, mural macrophage accumulation, and systolic blood pressure. The AAA suppression was also associated with reduced systemic levels of IL-1β but not IL-18. However, disulfiram treatment had no impact on body weight gain and lipid levels in aneurysmal mice. Additionally, disulfiram treatment also markedly reduced the secretion of IL-1β from activated macrophages with a limited effect on cell viability in vitro. Gasdermin D inhibition by disulfiram attenuated angiotensin II-induced experimental AAAs with reduced systemic IL-1β levels and in vitro activated macrophage IL-1β secretion. Our study suggests that pharmacological gasdermin D inhibition may have translational potential for limiting clinical AAA progression.

Targeting mitochondrial stress with SS31 prevents experimental abdominal aortic aneurysm: crosstalk with ER stress.

Background and purpose mitochondrial dysfunction and inflammation contribute to the pathophysiology of a myriad of cardiovascular diseases. Additionally, the deleterious crosstalk of mitochondria and persistent endoplasmic reticulum (ER) stress triggers oxidative stress, which is deeply involved in the development of vascular diseases. The purpose of this work was to determine whether inhibition of mitochondrial stress reduces aneurysm development in angiotensin II (Ang II)-infused apolipoprotein-E-deficient (ApoE-/- ) mice and its effect on ER stress. Experimental approach and results the mitochondria-targeted tetrapeptide SS31 ameliorated mitochondrial dysfunction and the enhanced expression of ER stress markers triggered by Ang II in ApoE-/- mice and limited plasmatic and vascular ROS levels. Interestingly, SS31 improved survival, reduced the incidence and severity of abdominal aortic aneurysm (AAA) and the AngII-induced increase of aortic diameter evaluated by ultrasonography, resembling the response triggered by the classic ER stress inhibitors TUDCA and PBA. The disorganization of extracellular matrix, the increased expression of metalloproteinases and pro-inflammatory markers and the infiltration of immune cells induced by AngII in the abdominal aorta were effectively reduced by both, SS31 and ER inhibitors. Further, CHOP deficiency in ApoE-/- mice attenuated the AngII-mediated increase in vascular diameter and the incidence of AAA, suggesting its contribution to the favorable response induced by ER stress inhibition. CONCLUSIONS: our data demonstrate that inhibition of mitochondrial stress by SS31 limits AAA formation and increases survival through a reduction of vascular remodeling, inflammation and ROS, and support that the attenuation of ER stress also contribute to the favorable response elicited by SS31.

ADAR1 Non-Editing Function in Macrophage Activation and Abdominal Aortic Aneurysm.

Macrophage activation plays a critical role in abdominal aortic aneurysm (AAA) development. However, molecular mechanisms controlling macrophage activation and vascular inflammation in AAA remain largely unknown. The objective of the study was to identify novel mechanisms underlying adenosine deaminase acting on RNA (ADAR1) function in macrophage activation and AAA formation. Aortic transplantation was conducted to determine the importance of nonvascular ADAR1 in AAA development/dissection. Ang II (Angiotensin II) infusion of ApoE-/- mouse model combined with macrophage-specific knockout of ADAR1 was used to study ADAR1 macrophage-specific role in AAA formation/dissection. The relevance of macrophage ADAR1 to human AAA was examined using human aneurysm specimens. Moreover, a novel humanized AAA model was established to test the role of human macrophages in aneurysm formation in human arteries. Allograft transplantation of wild-type abdominal aortas to ADAR1+/- recipient mice significantly attenuated AAA formation, suggesting that nonvascular ADAR1 is essential for AAA development. ADAR1 deficiency in hematopoietic cells decreased the prevalence and severity of AAA while inhibited macrophage infiltration and aorta wall inflammation. ADAR1 deletion blocked the classic macrophage activation, diminished NF-κB (nuclear factor kappa B) signaling, and enhanced the expression of a number of anti-inflammatory microRNAs. Mechanistically, ADAR1 interacted with Drosha to promote its degradation, which attenuated Drosha-DGCR8 (DiGeorge syndrome critical region 8) interaction, and consequently inhibited pri- to pre-microRNA processing of microRNAs targeting IKKβ, resulting in an increased IKKβ (inhibitor of nuclear factor kappa-B) expression and enhanced NF-κB signaling. Significantly, ADAR1 was induced in macrophages and interacted with Drosha in human AAA lesions. Reconstitution of ADAR1-deficient, but not the wild type, human monocytes to immunodeficient mice blocked the aneurysm formation in transplanted human arteries. Macrophage ADAR1 promotes aneurysm formation in both mouse and human arteries through a novel mechanism, that is, Drosha protein degradation, which inhibits the processing of microRNAs targeting NF-kB signaling and thus elicits macrophage-mediated vascular inflammation in AAA.

PS-BPB01-5: LOX-1 DEFICIENCY INCREASES RUPTURED ABDOMINAL AORTIC ANEURYSM VIA THINNING OF ADVENTITIAL COLLAGEN

Objective: Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a key mediator of inflammation and plays an important role in the pathogenesis of atherosclerosis. Both inflammation and atherosclerosis have been proposed as contributors to abdominal aortic aneurysm (AAA) pathogenesis. However, the role of LOX-1 in AAA pathogenesis remains unknown. We aimed to investigate effects of Olr1, which encodes LOX-1, deletion on angiotensin II (Ang II)-induced AAA in apolipoprotein E knockout (ApoE KO) mice to determine whether LOX-1 deficiency mitigates AAA development. Design and method: Either Ang II (1.44 mg/kg/day) or saline (control) was administered continuously through a subcutaneously implanted osmotic pump for 4 weeks in the following 8-week-old male mice: Apoe-/-Olr1+/+ mice (Ang II, n = 40; saline, n = 10), Apoe-/-Olr1+/- mice (Ang II, n = 30; saline, n = 9), and Apoe-/-Olr1-/- mice (Ang II, n = 39; saline, n = 9). Results: Ang II infusion raised systolic blood pressure by tail-cuff methods in all three groups. Serial non-invasive assessment with ultrasound demonstrated that Ang II gradually increased the maximum area of the suprarenal abdominal aortas leading to AAAs. Although the incidence and expansion rate of AAA were comparable among genotypes, Olr1 deletion significantly increased the severity and rupture rate of AAAs (p = 0.01; incidence of type IV and ruptured AAA was 25% in Apoe-/-Olr1-/- mice vs 5% in Apoe-/-Olr1+/+ mice). Since atherosclerosis is considered a major contributor to the development of AAAs, the extent of the atheroma burden localized in aneurysmal lesions was evaluated by Oil Red O staining. Olr1 deletion did not decrease atherosclerosis formation in the aneurysmal wall. Interestingly, further histopathological analysis revealed that aneurysmal lesions in LOX-1-deficient mice had fewer fibroblasts and myofibroblasts, as well as thinner adventitial collagen, although the degree of elastin fragmentation or disruption was similar between LOX-1-deficient and control mice. These in vivo data suggested that adventitial fibroblasts play a pivotal role in the increased severity of AAA in LOX-1-deficient mice. To further determine the effects of Olr1 deletion on fibroblast differentiation, fibroblasts were isolated from the adventitia of abdominal aortas harvested from 8-week-old Apoe-/-Olr1+/+ and Apoe-/-Olr1-/- mice, and then cultured with Ang II stimulation. An in vitro study confirmed that Olr1 deletion completely abolished the Ang II-induced proliferative activity in the aortic adventitial fibroblasts (p = 0.02). Conclusions: Olr1 deletion may not mitigate aneurysm development but rather increase the vulnerability to rupture by suppressing adventitial fibroblast proliferation and collagen.

The impact of neighborhood social disadvantage on abdominal aortic aneurysm severity and management

ObjectiveRecent studies have highlighted socioeconomic disparities in the severity and management of abdominal aortic aneurysm (AAA) disease. However, these studies focus on individual measures of social disadvantage such as income and insurance status. The area deprivation index (ADI), a validated measure of neighborhood deprivation, provides a more comprehensive assessment of social disadvantage. Therefore, we examined the impact of ADI on AAA severity and its management. MethodsWe identified all patients who underwent endovascular or open repair of an AAA in the Vascular Quality Initiative registry between 2003 and 2020. An ADI score of 1 to 100 was assigned to each patient based on their residential zip code, with higher ADI scores corresponding with increasing deprivation. Patients were categorized by ADI quintiles. Outcomes of interest included rates of ruptured AAA (rAAA) repair versus an intact AAA repair and rates of endovascular repair (EVAR) versus the open approach. Logistic regression was used to evaluate for an independent association between ADI quintile and these outcomes. ResultsAmong 55,931 patients who underwent AAA repair, 6649 (12%) were in the lowest ADI quintile, 11,692 (21%) in the second, 15,958 (29%) in the third, 15,035 (27%) in the fourth, and 6597 (12%) in the highest ADI quintile. Patients in the two highest ADI quintiles had a higher proportion of rAAA repair (vs intact repair) compared with those in the lowest ADI quintile (8.8% and 9.1% vs 6.2%; P < .001). They were also less likely to undergo EVAR (vs open approach) when compared with the lowest ADI quintile (81% and 81% vs 88%; P < .001). There was an overall trend toward increasing rAAA and decreasing EVAR rates with increasing ADI quintiles (P < .001). In adjusted analyses, when compared with patients in the lowest ADI quintile, patients in the highest ADI quintile had higher odds of rAAA repair (odds ratio, 1.4; 95% confidence interval, 1.2-1.8; P < .001) and lower odds of undergoing EVAR (odds ratio, 0.54; 95% confidence interval, 0.45-0.65; P < .001). ConclusionsAmong patients who underwent AAA repair in the Vascular Quality Initiative, those with higher neighborhood deprivation had significantly higher rates of rAAA repair (vs intact repair) and lower rates of EVAR (vs open approach). Further work is needed to better understand neighborhood factors that are contributing to these disparities to identify community-level targets for improvement.

Exosomal miR-17-5p from adipose-derived mesenchymal stem cells inhibits abdominal aortic aneurysm by suppressing TXNIP-NLRP3 inflammasome

BackgroundPreclinical studies have suggested that adipose-derived mesenchymal stem cells (ADSCs) transplantation can suppress abdominal aortic inflammation and aneurysm expansion through paracrine factors. Yet, the mechanism of action is not fully understood. In the present study, we further examined the function and mechanism of ADSC-derived exosomes (ADSC-exos) and their microRNA-17-5p (miR-17-5p) on the abdominal aortic aneurysm (AAA) progression.MethodsADSC-exos were isolated and identified. DiR and PKH67 staining were used to trace ADSC-exo in vivo and in vitro. Raw264.7 cells were applied to perform in vitro experiments, while a murine AAA model induced using angiotensin II (Ang II) was used for in vivo testing. The expression level of miR-17-5p in macrophages and Ang II-treated macrophages after ADSC-exos treatment was determined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The target relation between miR-17-5p and thioredoxin-interacting protein (TXNIP) was identified by a dual-luciferase reporter gene assay. Artificial activation and block of experiments of miR-17-5p and TXNIP were conducted to clarify their functions in inflammation during AAA progression. The severity of AAA between groups was assessed by maximal aorta diameter, AAA incidence, survival rate, and histological stainings. Besides, inflammasome-related proteins and macrophage pyroptosis were further evaluated using western blot, RT-qPCR, and enzyme-linked immunosorbent assay (ELISA).ResultsThe ADSC-exos were isolated and identified. In vivo testing showed that ADSC-exos were mainly distributed in the liver. Meanwhile, in vitro experiments suggested that ADSC-derived exosomes were taken up by macrophages, while inside, ADSC-exos miR-17-5p decreased a TXNIP induced by Ang II by directly binding to its 3′-untranslated region (3’UTR). Furthermore, overexpression of miR-17-5p enhanced the therapeutic function of ADSC-exos on inflammation during AAA expansion in vivo, while its inhibition reversed this process. Finally, overexpressed TXNIP triggered macrophage pyroptosis and was alleviated by ADSC-derived exosomes in vitro.ConclusionADSC-exos miR-17-5p regulated AAA progression and inflammation via the TXNIP-NLRP3 signaling pathway, thus providing a novel insight in 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.

Hemostatic Biomarkers and Volumetry Help to Identify High-Risk Abdominal Aortic Aneurysms.

Predicting the progression of small aneurysms is a main challenge in abdominal aortic aneurysm (AAA) management. The combination of circulating biomarkers and image techniques might provide an alternative for risk stratification. We evaluated the association of plasma TAT complexes (TAT) and D-dimer with AAA severity in 3 groups of patients: group 1, without AAA (n = 52), group 2, AAA 40–50 mm (n = 51) and group 3, AAA > 50 mm (n = 50). TAT (p < 0.001) and D-dimer (p < 0.001) were increased in patients with AAA (groups 2 and 3) vs. group 1. To assess the association between baseline TAT and D-dimer concentrations, and AAA growth, aortic diameter and volume (volumetry) were measured by computed tomography angiography (CTA) in group 2 at recruitment (baseline) and 1-year after inclusion. Baseline D-dimer and TAT levels were associated with AAA diameter and volume variations at 1-year independently of confounding factors (p ≤ 0.044). Additionally, surgery incidence, recorded during a 4-year follow-up in group 2, was associated with larger aneurysms, assessed by aortic diameter and volumetry (p ≤ 0.036), and with elevated TAT levels (sub-hazard ratio 1.3, p ≤ 0.029), while no association was found for D-dimer. The combination of hemostatic parameters and image techniques might provide valuable tools to evaluate AAA growth and worse evolution.

Abstract 350: Disruption Of Proprotein Convertase Subtilisin/Kexin 9 Suppresses Inflammation And Attenuates Abdominal Aortic Aneurysm Formation

Objective: Abdominal aortic aneurysm (AAA) is a degenerative inflammatory disease, with its rupture highly lethal. There have been no effective pharmacological medications. Proprotein convertase subtilisin/kexin 9 (PCSK9), which is known to modulate cholesterol metabolism, has recently been associated with AAA in human genome-wide association studies. However, its role in AAA pathogenesis is unknown. Approach and Results: Histology staining of mouse aortic aneurysmal tissue showed PCSK9 expression and its colocalization with macrophages. To investigate the role of PCSK9 in AAA formation, mice deficient in PCSK9 were crossed with ApoE SA/SA mice, which are hypercholesterolemic and inducible to develop angiotensin II-mediated hypertension upon doxycycline (Dox) administration. Deletion of PCSK9 reduced both incidence and severity of AAA and increased survival rate, without changing plasma cholesterol levels. This was associated with decreased aortic expression and activity of matrix metalloproteinase 9 (MMP9), less elastin breakdown, and reduced aortic expansion and aneurysm rupture. Additionally, PCSK9 deficiency suppressed systemic inflammatory response and macrophage infiltration into AAA lesions. In isolated macrophages stimulated with LPS, the upregulated expression of pro-inflammatory cytokines ( Tnfa, Il1b, Il6 ) was dramatically diminished in PCSK9-deficient macrophages. Furthermore, administration of PCSK9-specific siRNA in mice resulted in less macrophage accumulation in the aneurysmal aortic wall, decreased MMP9-mediated elastin degradation, and substantially constrained AAA formation. Conclusions: Endogenous PCSK9 promotes AAA formation, consistent with its novel action in mediating systemic and local inflammation, particularly, the macrophage-dependent MMP9 activity. Pharmacological inhibition of PCSK9 might be a promising strategy to curtail AAA. Key Words: aneurysm, PCSK9, elastin, inflammation, macrophages

Calcitriol Supplementation Protects Against Apoptosis and Alleviates the Severity of Abdominal Aortic Aneurysm Induced by Angiotensin II and Anti-TGFβ.

The present study aims to assess the effect of vitamin D deficiency (VDD) and its supplementation on the severity of AAA in mice. AAA was induced by AngII and anti-TGF-β administration. Animals were divided into four groups: Sham, mice with AAA, mice with AAA, and VDD, and mice with AAA supplemented with calcitriol. Blood pressure, echocardiography, abdominal aortic tissues, and plasma samples were monitored for all groups. VDD was associated with enhanced activity of cleaved MMP-9 and elastin degradation and positively correlated with the severity of AAA. Calcitriol supplementation decreased the INFγ/IL-10 ratio and enhanced the Nrf2 pathway. Moreover, Cu/Zn-superoxide dismutase expression and catalase and neutral sphingomyelinase activity were exacerbated in AAA and VDD groups. Furthermore, calcitriol supplementation showed a significantly lower protein expression of caspase-8, caspase-3, Bid, and t-Bid, and prevented the apoptosis of VSMCs treated by AngII and anti-TGF-β. Calcitriol supplementation may alleviate AAA severity and could be of great interest in the clinical management of AAA. VDD enhances antioxidant enzymes activity and expression, whereas calcitriol supplementation alleviates AAA severity by re-activating Nrf2 and inhibiting apoptotic pathways.

Pharmacological Inhibition of Factor XIIa Attenuates Abdominal Aortic Aneurysm, Reduces Atherosclerosis, and Stabilizes Atherosclerotic Plaques.

Background 3F7 is a monoclonal antibody targeting the enzymatic pocket of activated factor XII (FXIIa), thereby inhibiting its catalytic activity. Given the emerging role of FXIIa in promoting thromboinflammation, along with its apparent redundancy for hemostasis, the selective inhibition of FXIIa represents a novel and highly attractive approach targeting pathogenic processes that cause thromboinflammation-driven cardiovascular diseases. Methods The effects of FXIIa inhibition were investigated using three distinct mouse models of cardiovascular disease—angiotensin II-induced abdominal aortic aneurysm (AAA), an ApoE −/− model of atherosclerosis, and a tandem stenosis model of atherosclerotic plaque instability. 3F7 or its isotype control, BM4, was administered to mice (10 mg/kg) on alternate days for 4 to 8 weeks, depending on the experimental model. Mice were examined for the development and size of AAAs, or the burden and instability of atherosclerosis and associated markers of inflammation. Results Inhibition of FXIIa resulted in a reduced incidence of larger AAAs, with less acute aortic ruptures and an associated fibro-protective phenotype. FXIIa inhibition also decreased stable atherosclerotic plaque burden and achieved plaque stabilization associated with increased deposition of fibrous structures, a >2-fold thicker fibrous cap, increased cap-to-core ratio, and reduction in localized and systemic inflammatory markers. Conclusion Inhibition of FXIIa attenuates disease severity across three mouse models of thromboinflammation-driven cardiovascular diseases. Specifically, the FXIIa-inhibiting monoclonal antibody 3F7 reduces AAA severity, inhibits the development of atherosclerosis, and stabilizes vulnerable plaques. Ultimately, clinical trials in patients with cardiovascular diseases such as AAA and atherosclerosis are warranted to demonstrate the therapeutic potential of FXIIa inhibition.

Flow examination in abdominal aortic aneurysms: Reduced-order models driven by in vitro data and spectral proper orthogonal decomposition

Although flow characteristics in abdominal aortic aneurysms (AAAs) have been extensively studied, they have not found their way into the clinical evaluation of the disease. We, therefore, present experimental AAA flow models, optimized using spectral proper orthogonal decomposition, to aid in gauging AAA severity based on the underlying flow. The resulting reduced-order AAA flow models capture pertinent pathological flow features. With progressive aneurysm expansion, the flow becomes increasingly complex, accompanied by larger vortical structures and elevated particle residence time. The presented models can lead to a more comprehensive clinical evaluation that includes patient-specific flow information.

Dissecting Abdominal Aortic Aneurysm Is Aggravated by Genetic Inactivation of LIGHT (TNFSF14)

Abdominal aortic aneurysm (AAA), is a complex disorder characterized by vascular vessel wall remodeling. LIGHT (TNFSF14) is a proinflammatory cytokine associated with vascular disease. In the present study, the impact of genetic inactivation of Light was investigated in dissecting AAA induced by angiotensin II (AngII) in the Apolipoprotein E-deficient (Apoe−/−) mice. Studies in aortic human (ah) vascular smooth muscle cells (VSMC) to study potential translation to human pathology were also performed. AngII-treated Apoe−/−Light−/− mice displayed increased abdominal aorta maximum diameter and AAA severity compared with Apoe−/− mice. Notably, reduced smooth muscle α-actin+ area and Acta2 and Col1a1 gene expression were observed in AAA from Apoe−/−Light−/− mice, suggesting a loss of VSMC contractile phenotype compared with controls. Decreased Opn and augmented Sox9 expression, which are associated with detrimental and non-contractile osteochondrogenic VSMC phenotypes, were also seen in AngII-treated Apoe−/−Light−/− mouse AAA. Consistent with a role of LIGHT preserving VSMC contractile characteristics, LIGHT-treatment of ahVSMCs diminished the expression of SOX9 and of the pluripotency marker CKIT. These effects were partly mediated through lymphotoxin β receptor (LTβR) as the silencing of its gene ablated LIGHT effects on ahVSMCs. These studies suggest a protective role of LIGHT through mechanisms that prevent VSMC trans-differentiation in an LTβR-dependent manner.

Abstract MP23: Knockout Of Pgc1α In Endothelial Cells Augments Abdominal Aortic Aneurysm Formation

Introduction: It is well described that changes in the aortic media are crucial in the development of Abdominal Aortic Aneurysms (AAA), however the involvement of the endothelium is not as clear. The endothelium is the first vascular layer exposed to blood flow and aneurysms preferentially form in areas of disturbed flow. The regulatory mechanisms as to why aneurysms form at these sites remains unclear. Here we propose that the transcription coactivator peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) plays a pivotal role in the endothelium’s response to AAA formation in a shear responsive manner. Methods: 10-12 week old male mice deficient in endothelial specific PGC1α (KO) and their corresponding wild type background mice (WT) were treated with 0.75 mg/kg/day of Ang II. Our primary endpoints were measurement of the maximal width of the suprarenal aortas, frequency, and complexity based on the Daugherty classification. To determine the effects of shear, HAECs exposed to steady flow (15 dyn/cm 2 ) and disturbed flow (0±6 dyn/cm 2 at 1 Hz) for 12 hours were compared to determine if mRNA expression were different for PGC1α and SIRT1. Results: AAA incidence and severity were increased in ANG II-infused KO compared to the WT mice (100% vs. 20%, respectively). Measurements of excised aortas showed that KO mice exhibited larger AAA fold-increase compared to WT (2.00 ± 0.11 fold increase vs. 0.43 ± 0.19 fold increase, p-value 0.0002). The KO male mice also exhibited a more complex phenotype. HAECs that underwent disturbed flow had a significant decrease in expression of SIRT1 (p = 0.02) and trended towards a decrease in expression of PGC1α (p = 0.06). Conclusions: Our data strongly suggests that knockout of endothelial specific PGC1α leads to more frequent, complex and larger AAAs primarily in the suprarenal aorta, which is an area of disturbed flow. Interestingly, the addition of ANG II alone was able to induce AAA formation in our model. Additionally, we show that PGC1α and SIRT1 are regulated in a shear responsive manner. Taken together, endothelial PGC1α likely plays a regulatory role in AAA formation through shear-related mechanisms.

Erythropoietin promotes abdominal aortic aneurysms in mice through angiogenesis and inflammatory infiltration.

Abdominal aortic aneurysm (AAA) is a potentially fatal vascular disease, but the underlying mechanisms remain unknown. Here, we tested the hypothesis that erythropoietin (EPO) may promote the formation of AAA. We found that EPO dose-dependently promoted the formation of AAA in both Apoe -/- (66.7%) and wild-type (WT) (60%) mice receiving a high dose of EPO. EPO monoclonal antibodies given to Apoe -/- mice receiving angiotensin II (AngII) stimulation resulted in a markedly lower incidence of AAA (from 86.7 to 20%, P < 0.001), and EPO receptor (EPOR) knockdown in Epor +/- Apoe -/- mice substantially reduced the incidence of AAA compared to Apoe -/- mice after AngII stimulation (from 86.7 to 45.5%, P < 0.05), further supporting the finding that EPO is a contributor to AAA formation. EPO-induced AAA resulted in increased microvessels, phagocyte infiltration, and matrix metalloproteinase secretion, as well as reduced collagen and smooth muscle cells (SMCs). Experiments in vitro and ex vivo demonstrated that EPO induced proliferation, migration, and tube formation of endothelial cells via the JAK2/STAT5 signaling pathway. In humans, serum EPO concentrations were higher in patients with AAA than in healthy individuals and correlated with the size of the AAA, suggesting a potential link between EPO and the severity of AAA in humans. In conclusion, we found that EPO promotes the formation of AAA in both Apoe -/- and WT mice by enhancing angiogenesis, inflammation, collagen degradation, and apoptosis of SMCs and that EPO/EPOR signaling is essential for AngII-induced AAA. The association between EPO and AAA in humans warrants further study.