Pathophysiology Of Abdominal Aortic Aneurysm Research Articles

Metagenomic Analysis of Gut Microbiota for Abdominal Aortic Aneurysm.

Objectives: The pathophysiological mechanism of abdominal aortic aneurysm (AAA) remains unclear. We previously reported that Bifidobacterium adolescentis levels were reduced in the feces of patients with AAA by 16S ribosomal ribonucleic acid (RNA) gene sequencing. In this study, we increased the number of cases and conducted metagenomic analyses to examine bacterial genes associated with the pathophysiology of AAA. Methods: For gut microbiota data, feces from 55 patients with AAA and 52 patients with no history of AAA, lower extremity artery disease, or coronary artery disease (control group) were collected. Metagenomic analysis was performed by collecting raw stool samples from patients. For intestinal microbiota analysis, metagenomic analysis of the fecal samples was performed. Results: Oral bacteria, including Actinomyces oris (p <0.0001), Streptococcus salivarius (p <0.001), Lactobacillus salivarius (p <0.001), and Streptococcus sp. (p <0.001), were increased in the feces of patients with AAA. In addition, bacterial genes related to alpha lipoic acid (ALA) biosynthesis (M00882, M00883, and M00884, p <0.0001) were decreased in patients with AAA. Conclusions: In the feces of patients with AAA, there was an increase in oral bacteria, and the expression of bacterial genes related to ALA biosynthesis was reduced. The results suggest the possibility of developing gut microbial drug treatments for AAA.

Differential Expression Analyses on Human Aortic Tissue Reveal Novel Genes and Pathways Associated With Abdominal Aortic Aneurysm Onset and Progression.

Abdominal aortic aneurysms (AAAs) are focal dilatations of the abdominal aorta that expand progressively, increasing their risk of rupture. Rupture of an AAA is associated with high mortality rates, but the mechanisms underlying the initiation, expansion, and rupture of AAAs are not yet fully understood. We aimed to characterize the pathophysiology of AAAs and identify new genes associated with AAA initiation and progression. This study used RNA sequencing data on 140 samples, becoming the largest RNA sequencing data set for differential expression studies of AAAs. We performed differential expression analyses and analyses of differential splicing between dilated and nondilated aortic tissue samples, and between AAAs of different diameters. We identified 3002 differentially expressed genes between AAAs and controls that were independent of ischemic time, 1425 of which were new. Additionally, 8 genes (EXTL3, ZFR, DUSP8, DISP1, USP33, VPS37C, ZNF784, RFX1) were differentially expressed between AAAs of varying diameters and between AAAs and control samples. Finally, 7 genes (SPP1, FHL1, GNAS, MORF4L2, HMGN1, ARL1, RNASE4) with differential splicing patterns were also differentially expressed genes between AAAs and controls, suggesting that splicing differences in these genes may contribute to the observed expression changes and disease development. This study identifies new genes and splicing patterns associated with AAAs and validates previous relevant pathways on AAAs. These findings contribute to the understanding of the complex mechanisms underlying AAAs and may provide potential targets to limit AAA progression and mortality risk.

Insight in the (Phospho)proteome of Vascular Smooth Muscle Cells Derived From Patients With Abdominal Aortic Aneurysm Reveals Novel Disease Mechanisms.

Abdominal aortic aneurysm (AAA) is characterized by weakening and dilatation of the aortic wall in the abdomen. The aim of this study was to gain insight into cell-specific mechanisms involved in AAA pathophysiology by analyzing the (phospho)proteome of vascular smooth muscle cells derived from patients with AAA compared with those of healthy donors. A (phospho)proteomics analysis based on tandem mass spectrometry was performed on vascular smooth muscle cells derived from patients with AAA (n=24) and healthy, control individuals (C-SMC, n=8). Following protein identification and quantification using MaxQuant, integrative inferred kinase activity analysis was used to calculate kinase activity scores. Expression differences between vascular smooth muscle cells derived from patients with AAA and healthy, control individuals were predominantly found in proteins involved in ECM (extracellular matrix) remodeling (THSD4 [thrombospondin type-1 domain-containing protein 4] and ADAMTS1 [A disintegrin and metalloproteinase with thrombospondin motifs 1]), energy metabolism (GYS1 [glycogen synthase 1] and PCK2 [phosphoenolpyruvate carboxykinase 2, mitochondrial]), and contractility (CACNA2D1 [calcium voltage-dependent channel subunit α-2/δ-1] and TPM1 [tropomyosin α-1 chain]). Phosphorylation patterns on proteins related to actin cytoskeleton organization dominated the phosphoproteome of vascular smooth muscle cells derived from patients with AAA . Besides, phosphorylation changes on proteins related to energy metabolism (GYS1), contractility (PARVA [α-parvin], PPP1R12A [protein phosphatase 1 regulatory subunit 12A], and CALD1 [caldesmon 1]), and intracellular communication (GJA1 [gap junction α-1 protein]) were seen. Kinase activity of NUAK1 (NUAK family SNF1-like kinase 1), FYN (tyrosine-protein kinase Fyn), MAPK7 (mitogen-activated protein kinase 7), and STK10 (serine/threonine kinase 10) was different in vascular smooth muscle cells derived from patients with AAA compared with those from healthy, control individuals. This study revealed changes in expression and phosphorylation levels of proteins involved in various processes responsible for AAA progression and development (eg, energy metabolism, ECM remodeling, actin cytoskeleton organization, contractility, intracellular communication, and cell adhesion). These newly identified proteins, phosphosites, and related kinases provide further insight into the underlying mechanism of vascular smooth muscle cell dysfunction within the aneurysmal wall. Our omics data thereby offer the opportunity to study the relevance, either as drug target or biomarker, of these proteins in AAA development.

O64 ENHANCED EXPRESSION AND MEDIAL LAYER LOCALIZATION OF ELASTASE-2, AN ANGIOTENSIN ENZYME: IMPACT ON ABDOMINAL AORTIC ANEURYSM HALLMARKS IN MICE

Abdominal aortic aneurysm (AAA) is a pathological dilation of the abdominal aorta that can lead to death, with no pharmacological treatment available other than repair surgery. Continuous angiotensinII (AngII) infusion induces AAA in mice. Elastase-2 (ELA-2), a chymotrypsin-serine protease and an elastase family member, acts as an AngII enzyme in arteries and contributes to resistance arteries and vascular remodeling in animal models. Therefore, ELA-2 is a potential factor in AAA formation and/or development. We sought to investigate the expression and localization of ELA-2 in AAA-induced mice. Methods: Male wild-type (C57bl/6,WT) and ELA-2knockout (CELA-2aTm1Bdr; ELA-2KO) mice treated with saline (WT+Sal n=10, ELA-2KO+Sal, n=9) or AngII (1.500ng/kg/min, WT+AngII n=8, ELA-2KO+AngII n=8) for twenty-eight days by subcutaneously mini-pumps infusion. Vascular ultrasonography (US, Vevo2100) analyzed, before and after treatment, aortic expansion index, aortic systolic and diastolic diameter parameters. The immunofluorescence for ELA-2, aorta layer, and histologic staining to collagen, elastin, and fibrosis was analyzed. A correlation between ELA-2 and US was done. Results: WT+AngII mice developed AAA, and ELA-2KO mice did not have AAA by infusion of Ang II. Aorta ELA-2 expression up-regulated in WT-AngII vs. WT+Sal (p=0.0001). Aortic systolic diameter increased in the WT+AngII vs WT+Sal (0.8vs.1.5 mm, p&lt;0.0001). Aortic diastolic diameter increases in the WT+AngII vs WT+Sal (1.2 vs.0.6 mm, p&lt;0.0001). The aortic expansion index decreases in WT+Ang II vs WT+Sal (11.4 vs.17.1%, p=0.0008). WT+AngII aorta has higher collagen deposition and fibrosis and lower elastin than WT+Saline (p=0.001). ELA-2KO+AngII vs. ELA-2KO+SAL showed no difference in elastin, collagen, and fibrosis. The analyzed parameters characteristic of AAA showed a positive correlation with ELA-2 in aorta middle layer in WT+AngII, where AAA develops. Conclusion: ELA-2 expression is up-regulated in AAA mice’s aorta and may contribute to developing aneurysms. ELA-2KO might be less susceptible to developing AAA induced by AngII infusion. Therefore, ELA-2 plays a significant role in AAA pathophysiology and could be a potential target for therapeutic treatment to prevent AAA development.

Abstract 2128: Hepcidin Loss Demonstrates Sex Dependent Differences In Aortic Aneurysm Size

Background: Studies suggest circulating iron levels play a role in the pathophysiology of abdominal aortic aneurysms (AAAs). Low iron levels lead to medial degeneration and VSMC dysfunction while elevated iron levels promote oxidative stress. Hepcidin is a major iron regulatory hormone that binds ferroportin to reduce serum iron levels, and its removal in VSMCs has been shown to exacerbate AAAs; however, the role of sex in this mechanism remains understudied. The goal of the current study is to investigate the potential role of sex in iron metabolism in AAAs. Methods: Male and female C57BL/6 wild-type (WT/WT), heterozygous hepcidin knock-out (WT/KO) and hepcidin KO (KO/KO) underwent peri-adventitial application of porcine pancreatic elastase on the infrarenal aorta (n=10-19/group). At day 14, the aortas were measured using video micrometry and harvested for immunohistochemistry. In separate studies, male and female hepcidin WT/WT and KO/KO underwent elastase application and systemic b-aminopropionitrile (BAPN, 0.02%) administration and were harvested at 28 days. Finally, male and female LysM Cre Ferroportin flx/flx mice and their controls underwent elastase application and harvested at 14 days (n=5-10/group). Results: Hepcidin WT/KO and KO/KO male mice had dose dependent protection of aneurysm formation compared with controls (WT/WT: 145.5±33.8% vs WT/KO: 110.7±34.8% vs KO/KO: 53.7±13.7%, p=0.0001 by ANOVA). Female Hepcidin WT/KO and KO/KO mice exhibited increased AAA size compared with their controls (KO/KO: 119.0±23.5% vs WT/KO: 101.6 ±28.6% vs WT/WT: 71.2±22.0%, p =.0011 by ANOVA). In the elastase + BAPN model, Hepcidin KO/KO male mice had less aortic dilation compared to controls (KO/KO: 196.0 ±31.9% vs WT/WT: 476.8±82.2%, p =.0002 by Mann-Whitney) while female KO/KO mice exhibited greater aortic dilation (WT/WT: 186.7±26.0% vs. KO/KO: 406.9±62.2%, p=0.006). Male LysM Cre Ferroportin flx/flx mice had decreased aortic dilation (WT/WT: 132.5±19.3% vs flx/flx: 53.5±7.8%, p=0.001). Conclusion: Elimination of hepcidin in AAA formation exhibited protective effects in male and deleterious effects in female mice. This data suggests targeting of hepcidin using drug-based therapies could have gender-specific clinical implications.

Glycoprotein VI Platelet Receptor Blockade Attenuates Progression of Established Abdominal Aortic Aneurysms in Murine Models

Objective: Abdominal aortic aneurysms (AAA) frequently feature the formation of a nonocclusive intraluminal thrombus (ILT) along the site of aortic dilation. Platelets are known to maintain hemostasis and propagate thrombosis through several redundant activation mechanisms, yet the role of platelet activation in the pathogenesis of AAA associated ILT is still poorly understood. Thus, we sought to investigate how platelet activation via the glycoprotein VI receptor pathway impacts the pathogenesis of AAA. Methods and Results: RNA sequencing was performed on age-matched control human aortic tissue, AAA aortic tissue, and AAA ILT. Platelet transcripts comprised 25% of significantly enriched genes in the comparison of AAA thrombus to AAA tissue and control aortic wall, with GPVI increased by 9.1 Log2-FC (P = 3.21E−08). Circulating GPVI was significantly elevated in platelets isolated from AAA patients versus age-matched controls. Soluble GPVI (sGPVI—indicator of platelet activation), was significantly increased in the plasma of patients with fast-growing AAAs compared to slow-growing AAAs and healthy control subjects. To determine if GPVI blockade was protective from AAA progression, mice underwent either the angiotensin II (AngII) infusion or the topical elastase models of aneurysm formation. Following model initiation, aortic diameter was monitored by weekly ultrasounds. After two weeks, mice which developed an aneurysm (defined as an aortic diameter of &gt;1.2mm) were randomized into control (IgG—50μg) or treatment (JAQ1—50μg, monoclonal GPVI antibody) groups. As expected, platelets isolated from JAQ1 treated mice did not activate in response to the GPVI specific agonist convulxin. Furthermore, JAQ1 treatment attenuated aneurysm progression, increased type I collagen deposition, attenuated macrophage accumulation, and increased survival in both murine AAA models versus IgG control treated mice. Conclusions: Platelets play a critical role in AAA pathophysiology and specific blockade of the GPVI-mediated activation pathway attenuates progression of established aneurysms in two independent murine models. Thus, our work demonstrates that specific targeting of GPVI to blunt platelet activation in AAA could be a potential therapeutic for a pathology with no current pharmacological treatments. R01HL147171-04 and R01HL158801-02. 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 3002: Pcsk9 In Abdominal Aortic Aneurysm - Beyond Hypercholesterolemia

Background: Our lab has identified a role for proprotein convertase subtilisin/kexin type9 (Pcsk9) in murine abdominal aortic aneurysm (AAA). Beyond its pathological effects on degradation of LDL-receptors, and enhancement of tissue inflammation via the NF-kB pathway, reports suggest that it alters vascular smooth muscle cell (VSMC) senescence and metabolism. However, the specific cellular mechanisms connecting Pcsk9 to AAA disease (particularly SMC-derived Pcsk9) are still unclear. Hypothesis: Intercellular communication between VSMCs and macrophages via enhanced local Pcsk9 expression drives macrophages towards pro-inflammatory polarization and promotes AAA growth by upregulated aortic wall inflammation. Pcsk9 also enhances mitochondrial metabolism in VSMCs, contributing to cell senescence. Pcsk9 knockdown inhibits these effects. Aims: We explored the impacts of SMC-derived Pcsk9 on aortic inflammation and VSMC behavior, seeking to further understand AAA pathophysiology and provide a basis for re-purposing Pcsk9 inhibitors as a treatment for non-surgical AAA. Methods: Single-cell RNASeq of murine AAA tissue (porcine pancreatic elastase model) utilizing control and Pcsk9-knockout mice (systemic) was performed to examine local changes in aortic wall cell-subtype gene expression. Pcsk9 silenced VSMCs (with siRNA) were co-cultured with macrophages to assess polarization towards pro-inflammatory phenotype and pro-inflammatory gene/protein expression was identified. VSMCs were also treated either with exogenous Pcsk9 or Pcsk9 siRNA in vitro to assess mitochondrial metabolism via Seahorse assay. Results: Pcsk9 knock-out AAA tissue exhibited reductions in pro-inflammatory gene expression profiles in vascular cell sub-populations (compared to wildtype). In vitro, Pcsk9-silenced VSMC-cocultured macrophages presented decreased inflammatory profile assessed by flow cytometry. Further, exogenous Pcsk9-treated-VSMCs displayed metabolic changes compared to control (Mann-Whitney Test). Conclusions: Our data suggest significant mechanistic involvement of Pcsk9 in AAA disease at the cellular level. Absence of Pcsk9 appears to mitigate vascular inflammation and alter VSMC metabolism in ways that may decelerate AAA.

Abstract 2014: Long Non-coding RNA LINC-PINT Regulates Smooth Muscle Cell Plasticity In Developing Abdominal Aortic Aneurysms

Background: Long noncoding RNAs (lncRNAs) play pivotal roles as molecular regulators in diverse vascular biological processes and diseases. This study utilized multi-transcriptomic profiling to investigate the contribution of lncRNAs in Abdominal Aortic Aneurysm (AAA) disease. Materials &amp; Methods: Utilizing bulk and single-cell RNA sequencing, we identified lncRNA long intergenic non-protein coding RNA, p53 induced transcript (LINC-PINT) as a significantly deregulated transcript in AAA compared to non-dilated aortas. Deconvolution of single-cell and bulk RNA-sequencing via the BayesPrism tool identified plasticity of smooth muscle cells (SMCs) as the predominant shifted condition between dilated vs. non-dilated aortas. Hybridization-based RNA in situ sequencing (HybRISS) confirmed the presence of LINC-PINT in de-differentiating SMCs. Results: Experimental knock-down of LINC-PINT , using site-specific antisense oligonucleotides (LNA-GapmeRs) markedly decreased proliferation rates of cultured human aortic smooth muscle cells (AoSMCs), but also increased their apoptosis, whereas overexpression of LINC-PINT had the opposite effect. Transcriptomic profiling of LINC-PINT -silenced AoSMCs indicated down-regulation of contractility markers, accompanied by an increase of inflammation and extracellular matrix (ECM) degradation. In line with this, LINC-PINT knock-down abolished the transforming growth factor beta (TGF-β)- induced AoSMCs polarization towards a contractile phenotype. Notably, RNA immunoprecipitation (RIP) demonstrated an interaction between LINC-PINT and p65, inhibiting its translocation to the promoter region of inflammatory genes. LINC-PINT knock-down increased p65 binding to its coactivator p50, which was confirmed by Co-immunoprecipitation (CO-IP). Combined in silico prediction and luciferase reporter assays further revealed that LINC-PINT expression was controlled by the ZNF93 and ZNF263 transcription factors. Conclusion: We provide evidence of LINC-PINT being a novel regulator influencing SMC dynamics and fate decisions of central importance to AAA pathophysiology. Currently ongoing in vivo studies in Lncpint -deficient mice explore its relevance in experimental AAA development.

Abstract 1010: Novel Murine Model For Infrarenal Abdominal Aortic Aneurysm Development And Rupture

Introduction: Abdominal Aortic Aneurysm (AAA) rupture is linked with increased mortality. Multiple rodent models attempt to capitulate AAA pathology to investigate a preventative cure. However, there are currently no murine models that reliably mimic AAA rupture. Compounds such as porcine pancreatic elastase (PPE), papain, β-aminopropionitrile (BAPN) and angiotensin II (ANG II) can independently promote AAA formation in rodents. We hypothesize that combining these agents can lead to reliable infrarenal AAA rupture model in mice. Methods: 28 male C57BL/6 mice underwent aortic topical exposure to 50μL of either saline (sham), PPE, papain or both PPE+papain (20 &amp; 10.3mg/mL respectively) for 20 minutes with a cotton ball to induce AAA degeneration. A group of 10 mice were also exposed to BAPN at 0.3% via drinking water (Fig 1A). After 3 days, another group of 5 mice was further exposed to ANG II subcutaneous pump. Weight and in vivo aortic size were measured longitudinally. Mice that died spontaneously were promptly assessed to evaluate for AAA rupture (Fig 1B). All remaining mice were sacrificed at week 4. Results: Compared to sham, mice exposed to papain or PPE+papain, but no BAPN or ANG II, demonstrated no change in weight gain, but significantly increased aortic diameter at week 4 (p&lt;0.01) while administration of 0.3% BAPN demonstrated further increase in AAA diameter in both groups (p=0.002 and p&lt;0.001, respectively; Fig 1C). All mice that received PPE+papain+0.3% BAPN as well as ANG II pump developed infrarenal AAAs and 80% developed a free rupture by day 9 (Fig 1D). Conclusion: We present a novel murine model that provides a high rate of infrarenal AAA development and rupture. The model not only combines matrix degradation, but also impulse risk factors known to enhance AAA rupture in humans. The feasibility and reliability of the model facilitates its use in future murine genetic and pharmacological studies that aim to understand AAA pathophysiology and methods of prevention.

Integrated particle image velocimetry and fluid–structure interaction analysis for patient-specific abdominal aortic aneurysm studies

BackgroundUnderstanding the hemodynamics of an abdominal aortic aneurysm (AAA) is crucial for risk assessment and treatment planning. This study introduces a low-cost, patient-specific in vitro AAA model to investigate hemodynamics using particle image velocimetry (PIV) and flow-simulating circuit, validated through fluid–structure interaction (FSI) simulations.MethodsIn this study, 3D printing was employed to manufacture a flexible patient-specific AAA phantom using a lost-core casting technique. A pulsatile flow circuit was constructed using off-the-shelf components. A particle image velocimetry (PIV) setup was built using an affordable laser source and global shutter camera, and finally, the flow field inside the AAA was analyzed using open-source software. Fluid–structure interaction (FSI) simulations were performed to enhance our understanding of the flow field, and the results were validated by PIV analysis. Both steady-state and transient flow conditions were investigated.ResultsOur experimental setup replicated physiological conditions, analyzing arterial wall deformations and flow characteristics within the aneurysm. Under constant flow, peak wall deformations and flow velocities showed deviations within − 12% to + 27% and − 7% to + 5%, respectively, compared to FSI simulations. Pulsatile flow conditions further demonstrated a strong correlation (Pearson coefficient 0.85) in flow velocities and vectors throughout the cardiac cycle. Transient phenomena, particularly the formation and progression of vortex structures during systole, were consistently depicted between experimental and numerical models.ConclusionsBy bridging high-fidelity experimental observations with comprehensive computational analyses, this study underscores the potential of integrated methodologies in enhancing our understanding of AAA pathophysiology. The convergence of realistic AAA phantoms, precise PIV measurements at affordable cost point, and validated FSI models heralds a new paradigm in vascular research, with significant implications for personalized medicine and bioengineering innovations.

Abstract 17672: Implications of Pcsk9 on Abdominal Aortic Aneurysm

Objective: Recent genome-wide association studies revealed a potential role of proprotein convertase subtilisin/kexin type9(Pcsk9) in the human abdominal aortic aneurysm(AAA) disease.Altered Pcsk9 gene expression and smaller AAA diameter were observed in murine AAA using the porcine pancreatic elastase(PPE) model in our lab. Pcsk9 increases plasma LDL-cholesterol,enhances tissue inflammation and induces vascular smooth muscle cell (VSMC) senescence, hallmarks in AAA.However, the exact cellular mechanisms of Pcsk9 in AAA disease remain unclear.Despite the absence of a pharmacological treatment for AAA, monoclonal Pcsk9 antibody therapies have proven effective against statin-resistant hypercholesterolemia.This study aimed to explore the impacts of Pcsk9 on aortic vascular inflammation and VSMC dedifferentiation with a view to better understanding AAA pathophysiology and providing a basis for future investigations into Pcsk9 inhibitors. Hypothesis: We hypothesized that local vascular injury escalates Pcsk9 expression,amplifying vascular inflammation and driving VSMC towards dedifferentiation that further advances AAA. Conversely,Pcsk9 inhibition will mitigate these processes. Methods: AAA was induced by PPE model in wildtype C57Bl/6 mice and Pcsk9 knock-out (KO)mice.Sonography was used to evaluate the aneurysm growth. Single-cell RNA sequencing was employed to examine local changes in the aortic wall. Local vascular Pcsk9,pro-inflammatory and VSMC marker gene/protein expression were quantified using qPCR and immunohistochemistry.VSMCs were treated with exogenous Pcsk9 in vitro to assess metabolism via Seahorse. Results: Reduced AAA diameters were observed in Pcsk9 KO mice compared to wildtype controls. Significant upregulation of local Pcsk9 expression accompanied AAA induction. Single-cell RNA sequencing revealed altered gene expression profiles across vascular cell populations. In vitro,VSMCs displayed changes in marker gene/protein expression after treatment with exogenous Pcsk9, alongside metabolic changes (Mann-Whitney Test). Conclusions: The findings suggest a significant role of Pcsk9 in AAA pathogenesis. Its inhibition appears to mitigate AAA growth,indicating a promising treatment target for AAA.

Assessing Efficacy of Afatinib toward Elastic Matrix Repair in Aortic Aneurysms.

Abdominal aortic aneurysm (AAA) is a critical, multifactorial cardiovascular disorder marked by localized dilatation of the abdominal aorta. A major challenge to countering the pathophysiology of AAAs lies in the naturally irreversible breakdown of elastic fibers in the aorta wall, which is linked to the poor elastogenicity of adult and diseased vascular smooth muscle cells (SMCs) and their impaired ability to assemble mature elastic fibers in a chronic proteolytic tissue milieu. We have previously shown that these are downstream effects of neutrophil elastase-induced activation of the epidermal growth factor receptor (EGFR) activity in aneurysmal SMCs. The novelty of this study lies in investigating the benefits of an EGFR inhibitor drug, afatinib (used to treat nonsmall cell lung cancer), for proelastogenic and antiproteolytic stimulation of aneurysmal SMCs. In in vitro cell cultures, we have shown that safe doses of 0.5 and 1 nM afatinib inhibit EGFR and p-extracellular signal-regulated kinases 1/2 protein expression by 50-70% and downstream elastolytic matrix metalloprotease 2 (MMP2) versus untreated control cultures. In addition, elastin production on a per cell basis was significantly upregulated by afatinib doses within the 0.1-1 nM dose range, which was further validated through transmission electron microscopy showing significantly increased presence of tropoelastin coacervates and maturing elastic fibers upon afatinib treatment at the above doses. Therefore, our studies for the first time demonstrate the therapeutic benefits of afatinib toward use for elastic matrix repair in small AAAs.

Regional and Global Aortic Pulse Wave Velocity in Patients with Abdominal Aortic Aneurysm

Abdominal aortic aneurysm (AAA) is commonly defined as a localised dilatation with a diameter > 30 mm. The pathophysiology of AAA includes chronic inflammation and enzymatic degradation of elastin, possibly increasing aortic wall stiffness and pulse wave velocity (PWV). Whether aortic stiffness is more prominent in the abdominal aorta at the aneurysm site is not elucidated. The aim of this study was to evaluate global and regional aortic PWV in patients with AAA. Experimental study of local PWV in the thoracic descending and abdominal aorta in patients with AAA and matched controls. The study cohort comprised 25 patients with AAA > 30 mm (range 36 - 70 mm, all male, age range 65 - 76 years) and 27 age- and sex-matched controls free of AAA. PWV was measured with applanation tonometry (carotid-femoral PWV, cfPWV) as well as 4D flow MRI technique, assessing regional aortic PWV. Blood pressure and anthropometrics were measured. Global aortic PWV was greater in men with AAA than controls, both by MRI (AAA 8.9 ± 2.4 m/s vs. controls 7.1 ± 1.5 m/s; p = .007) and cfPWV (AAA 11.0 ± 2.1 m/s vs. controls 9.3 ± 2.3 m/s; p = .007). Regionally, PWV was greater in the abdominal aorta in the AAA group (AAA 7.0 ± 1.8 m/s vs. controls 5.8 ± 1.0 m/s; p = .022), but similar in the thoracic descending aorta (AAA 8.7 ± 3.2 m/s vs. controls 8.2 ± 2.4 m/s; p = .59). Furthermore, PWV was positively associated with indices of central adiposity both in men with AAA and controls. PWV is higher in men with AAA compared with matched controls in the abdominal but not the thoracic descending aorta. Furthermore, aortic stiffness was linked with central fat deposition. It remains to be seen whether there is a causal link between AAA and increased regional aortic stiffness.

ROLE OF METALLOPROTEINASES AND ROS GENERATION IN THE AORTA OF ELASTASE-2 KNOCKOUT MICE IN ABDOMINAL AORTIC ANEURYSM MODEL

Objective: Elastase-2 (ELA-2) enzyme generates angiotensin-II (AngII) in arteries and is implicated in abnormal vascular remodeling in animal models. Our group demonstrated previously that ELA-2 knockout (ELA-2KO) mice were less susceptible to developing an aorta dilation in response to AngII infusion. This study hypothesizes that metalloproteinases and ROS generation in ELA-2KO mice mediate less susceptibility to aorta dilation and abdominal aortic aneurysm (AAA). Design and method: Male wild type (C57bl/6, WT) and ELA-2KO (CELA-2aTm1Bdr) mice were treated with either saline or AngII (2.28g/g/min) for twenty-eight days by subcutaneously mini-pumps infusion. Mice were divided into four groups: WT treated with saline (WT+SAL, n = 5), ELA-2KO treated with saline (ELA-2KO+SAL, n = 5), WT treated with AngII (WT+AngII,n = 5), and ELA-2KO treated with AngII (ELA-2KO+AngII, n = 5). The vascular ultrasound (US) was done before treatment and once a week during 28 days of treatment. The MMP- 2 and MMP-9 analyzes were done by zymography, ROS generation by DHE analyses, and alfa-actin expression by immunofluorescence in all aorta mice groups. ANOVA and Kruskal Wallis tests were used to compare the average between groups. This study is approved by CEUA (n.131/2019). Results: AngII treatment was associated with increased MMPs expression in the WT group aorta (WT+AngII vs. WT+SAL p&lt;0.05). The aorta ELA-2KO group showed no difference in MMPs in response to AngII infusion compared to ELA-2KO+SAL group (p = 0.81). The ROS generation had upregulated in aorta ELA-2KO+Ang II mice compared to ELA-2KO+SAL (p&lt;0.001) and aorta WT+AngII compared with WT+SAL (p&lt;0.0001). AngII treatment was associated with decreased ROS generation in the aorta ELA-2KO+AngII group compared with WT+AngII (p = 0.0051). The alfa-actin expression was similar in all groups. Our data demonstrate that AngII treatment did not increase the expression of MMPs in aorta ELA-2KO but was associated with an upregulation for ROS generation. Conclusions: Taken together, this suggests that ELA-2KO mice's resistance to developing an aorta dilation/AAA is related to MMPs and ROX generation. Our results also highlight the importance of the local/tissue response in the pathophysiology of AAA.

Mitochondrial Dysfunction and Increased DNA Damage in Vascular Smooth Muscle Cells of Abdominal Aortic Aneurysm (AAA-SMC).

There is increasing evidence for enhanced oxidative stress in the vascular wall of abdominal aortic aneurysms (AAA). Mitochondrial damage and dysfunction are hypothesized to be actors in altered production of reactive oxygen species (ROS) and oxidative stress. However, the role of mitochondria and oxidative stress in vascular remodelling and progression of AAA remains uncertain. We here addressed whether mitochondrial dysfunction is persistently increased in vascular smooth muscle cells (VSMCs) isolated from AAA compared to healthy VSMC. AAA-derived VSMC cultures (AAA-SMC, n = 10) and normal VSMC cultures derived from healthy donors (n = 7) were grown in vitro and analysed for four parameters, indicating mitochondrial dysfunction: (i) mitochondrial content and morphology, (ii) ROS production and antioxidative response, (iii) NADP+/NADPH content and ratio, and (iv) DNA damage, in the presence or absence of angiotensin II (AngII). AAA-SMC displayed increased mitochondrial circularity (rounded shape), reduced mitochondrial area, and reduced perimeter, indicating increased fragmentation and dysfunction compared to healthy controls. This was accompanied by significantly increased O2 - production, reduced NADP+/NADPH levels, a lower antioxidative response (indicated by antioxidative response element- (ARE-) driven luciferase reporter assays), more DNA damage (determined by percentage of γ-H2A.X-positive nuclei), and earlier growth arrest in AAA-SMC. Our data suggest that mitochondrial dysfunction and oxidative stress are persistently increased in AAA-SMC, emphasizing their implication in the pathophysiology of AAA.

Abdominal Aortic Aneurysm: Natural History, Pathophysiology and Translational Perspectives.

An abdominal aortic aneurysm (AAA) is a degenerative pathology that affects the infrarenal segment of the aorta, leading to its progressive dilatation and eventually rupture. The infrarenal segment is involved in 80% of the aortic aneurisms, and represents alone 30% of all aneurysms. The natural history of the disease is characterized by the progressive increase of the aortic diameter associated with proportionally higher risk of rupture, particularly for cases with diameter greater than 5.5 cm. In case of rupture the mortality rate is very high, independently from the endovascular or surgical treatment. The most important risk factors are older age, smoking, hypertension, dyslipidemia, and family history of AAA. The most frequent form is "atherosclerotic", but infectious, collagen disease-related, immune dysregulation-related, and post-traumatic AAA have also been described. Albeit multiple pathogenetic hypotheses have been proposed, the role of metallo-proteinases in the degeneration of the aortic wall seem to play a central role. Early detection of AAA is crucial for the identification and treatment before the onset of potentially life-threatening complications. Moreover, the individual risk stratification is fundamental for the clinical management and follow-up. The growing knowledge about the pathophysiology of AAA has the potential to lead to significant translational advances. The challenge for the next years is to employ bioinformatic and genetic models, also based on artificial intelligence and machine learning approach, to develop novel screening methods and to stratify individuals at higher-risk or in the early stages of AAA.

Gut microbiome sheds light on the development and treatment of abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) is an inflammatory vascular disease with high disability and mortality. Its susceptible risk factors include old age, being male, smoking, hypertension, and aortic atherosclerosis. With the improvement of screening techniques, AAA incidence and number of deaths caused by aneurysm rupture increase annually, attracting much clinical attention. Due to the lack of non-invasive treatment, early detection and development of novel treatment of AAA is an urgent clinical concern. The pathophysiology and progression of AAA are characterized by inflammatory destruction. The gut microbiota is an "invisible organ" that directly or indirectly affects the vascular wall inflammatory cell infiltration manifested with enhanced arterial wall gut microbiota and metabolites, which plays an important role in the formation and progression of AAA. As such, the gut microbiome may become an important risk factor for AAA. This review summarizes the direct and indirect effects of the gut microbiome on the pathogenesis of AAA and highlights the gut microbiome-mediated inflammatory responses and discoveries of relevant therapeutic targets that may help manage the development and rupture of AAA.

Abstract P114: Differential Expression Of Angiotensin Ii Receptors In Aorta Of Ela2ko Mice With Abdominal Aortic Aneurysm.

Elastase-2 (ELA-2) enzyme generates angiotensin-II (AngII) in arteries and is implicated in abnormal vascular remodeling in animal models.Our group demonstrated previously that ELA-2 knockout (ELA-2KO) mice were less susceptible to develop an aorta dilation in a response to AngII infusion. In this study, we hypothesize that susceptibility to an aorta dilation and abdominal aortic aneurysm (AAA) is mediated by AngII receptors (AT1 and AT2). Methods: Male wild type (C57bl/6, WT) and ELA-2KO (CELA-2aTm1Bdr) mice were treated with either saline or AngII (2.28g/g/min) for twenty-eight days by subcutaneously mini-pumps infusion. Mice were divided into four groups: WT treated with saline (WT+SAL, n=5), ELA-2KO treated with saline (ELA-2KO+SAL, n=5), WT treated with AngII (WT+AngII,n=5), and ELA-2KO treated with AngII (ELA-2KO+AngII, n=5). Protein expression of AT1 and AT2 receptor, CD68, and α-actin was measured by immunofluorescence in formalin-fixed, paraffin-embedded aorta tissue. ANOVA and Kruskal Wallis tests were used to compare the average fluorescent intensity between groups. Results: AngII treatment was associated with increased AT1 receptor expression in a WT group, but not with AT2 receptor expression (vs. WT+SAL,p=0.06 and p=0.31, respectively). The ELA-2KO group showed no difference in AT1 receptor expression in a response to AngII infusion compared to a ELA-2KO+SAL group (p=0.81). While ELA-2KO+Ang II mice had lower AT2 expression compared to ELA-2KO+SAL (p=0.05). The α-actin expression was similar in all groups. WT+AngII group expressed more CD68 expression in a response to an AngII treatment that was increased in a WT group (p=0.03), but not in a ELA-2KO group. Conclusion: Our data demonstrate that Ang II treatment did not increase an expression of AT1 receptor in ELA-2KO, but was associated with a downregulation of AT2 receptors. Taken together, this suggests That ELA-2KO mice resistance to developing an aorta dilation/AAA is related to downregulation of Ang II receptors. Our results also highlight the importance of the local/tissue response in the pathophysiology of AAA.

Progress in murine models of ruptured abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) is a focal dilation of the aorta that is prevalent in aged populations. The progressive and unpredictable expansion of AAA could result in aneurysmal rupture, which is associated with ~80% mortality. Due to the expanded screening efforts and progress in diagnostic tools, an ever-increasing amount of asymptomatic AAA patients are being identified yet without a cure to stop the rampant aortic expansion. A key barrier that hinders the development of effective AAA treatment is our incomplete understanding of the cellular and molecular basis of its pathogenesis and progression into rupture. Animal models provide invaluable mechanistic insights into AAA pathophysiology. However, there is no single experimental model that completely recapitulate the complex biology behind AAA, and different AAA-inducing methodologies are associated with distinct disease course and rupture rate. In this review article, we summarize the established murine models of ruptured AAA and discuss their respective strengths and utilities.

Adult Mesenchymal Stem Cells and Derivatives in Improved Elastin Homeostasis in a Rat Model of Abdominal Aortic Aneurysms.

Abdominal aortic aneurysms (AAAs) are localized rupture-prone expansions of the aorta with limited reversibility that develop due to proteolysis of the elastic matrix. Natural regenerative repair of an elastic matrix is difficult due to the intrinsically poor elastogenicity of adult vascular smooth muscle cells (VSMCs). This justifies the need to provide external, pro-elastin regenerative- and anti-proteolytic stimuli to VSMCs in the AAA wall towards reinstating matrix structure in the aorta wall. Introducing alternative phenotypes of highly elastogenic and contractile cells into the AAA wall capable of providing such cues, proffers attractive prospects for AAA treatment. In this regard, we have previously demonstrated the superior elastogenicity of bone marrow mesenchymal stem cell (BM-MSC)-derived SMCs (cBM-SMCs) and their ability to provide pro-elastogenic and anti-proteolytic stimuli to aneurysmal SMCs in vitro. However, the major issues associated with cell therapy, such as their natural ability to home into the AAA tissue, their in vivo biodistribution and retention in the AAA wall, and possible paracrine effects on AAA tissue repair processes in the event of localization in remote tissues remain uncertain. Therefore, in this study we focused on assessing the fate, safety, and AAA reparative effects of BM-MSC-derived cBM-SMCs in vivo. Our results indicate that the cBM-SMCs (a) possess natural homing abilities similar to the undifferentiated BM-MSCs, (b) exhibit higher retention upon localization in the aneurysmal aorta than BM-MSCs, (c) downregulate the expression of several inflammatory and pro-apoptotic cytokines that are upregulated in the AAA wall contributing to accelerated elastic matrix breakdown and suppression of elastic fiber neo-assembly, repair, and crosslinking, and (d) improve elastic matrix content and structure in the AAA wall toward slowing the growth of AAAs. Our study provides initial evidence of the in vivo elastic matrix reparative benefits of cBM-SMCs and their utility in cell therapy to reverse the pathophysiology of AAAs.