Experimental Models Of Abdominal Aortic Aneurysm Research Articles

CD8+ T-cell deficiency protects mice from abdominal aortic aneurysm formation in response to calcium chloride2.

Abdominal aortic aneurysm (AAA) is an aneurysm-like dilated and highly fatal cardiovascular disease. CD8+ T cells have been shown to be critical for vascular pathological processes, but the contribution of these lymphocytes to vascular diseases remains elusive. Eight-week-old male wildtype (CD8+/+) and Cd8a knockout (CD8-/-) mice were used in a calcium chloride2 (CaCl2)-induced experimental AAA model. At 6 weeks after surgery, CD8+ T-cell deletion prevented the formation of AAA, accompanied by reductions of the levels of inflammatory (interferon-γ [IFN-γ], interleukin-1β, monocyte chemoattractant protein-1, intracellular adhesion molecule-1, vascular cell adhesion molecule-1, NOD-like receptor protein 3, caspase-1), oxidative stress [NADPH oxidase and gp91phox], and proteolysis (cathepsin S, cathepsin K, matrix metalloproteinase-2 [MMP-2] and MMP-9) proteins and/or genes in plasma and/or AAA tissues. Immunoreactivities of MMP-2 and MMP-9 were observed in macrophages. An injection of IFN-γ and adoptive transfer of CD8+ T cells of IFN-γ+/+ mice diminished CD8-/--mediated vasculoprotective actions in the AAA mice. In vitro, IFN-γ enhanced MMP-2 and MMP-9 gelatinolytic activities in macrophage and/or vascular smooth muscle cells. The vasculoprotective effects of CD8+ T-cell deletion in a mouse CaCl2-induced AAA model were likely attributable to, at least in part, the attenuation of IFN-γ-dependent inflammation action, oxidative stress production, and proteolysis, suggesting a novel therapeutic target for AAA formation by regulating CD8+ T-cell-derived IFN-γ secretion.

Mouse Abdominal Aortic Aneurysm Model Induced by Periarterial Incubation of Papain

For decades, numerous experimental animal models have been developed to examine the pathophysiologic mechanisms and potential treatments for abdominal aortic aneurysms (AAAs) in diverse species with varying chemical or surgical approaches. This study aimed to create an AAA mouse model by the periarterial incubation with papain, which can mimic human AAA with advantages such as simplicity, convenience, and high efficiency. Eighty C57BL/6J male mice were randomly assigned to 1 of the 4 groups: papain (1.0 or 2.0 mg), porcine pancreatic elastase, and phosphate-buffered solution. The aortic segment was wrapped for 20 minutes, and the diameter was measured using ultrasound preoperatively and postoperative days 7 and 14. Then, the mice were killed for histomorphometric and immunohistochemical analyses. According to ultrasound measurements and histomorphometric analyses, on postoperative day 7, 65% of mice in the 1.0-mg papain group and 60% of mice in the 2.0-mg papain group developed AAA. In both papain groups, 100% of mice developed AAA, and 65% of mice in the porcine pancreatic elastase group developed AAA on postoperative day 14. Furthermore, hematoxylin/eosin, elastin van Gieson, and Masson staining of tissues from the papain group revealed thickened media and intimal hyperplasia, collagen sediments, and elastin destruction, indicating that AAA histochemical alteration was similar to that of humans. In addition, the immunohistochemical analysis was conducted to detect infiltrated inflammatory cells, such as macrophages and leukocytes, in the aortic wall and hyperplasic adventitia. The expression of matrix metalloproteinase 2 and 9 was significantly upregulated in papain and human AAA tissues. Periarterial incubation with 1.0 mg of papain for 20 minutes can successfully create an experimental AAA model in mice for 14 days, which can be used to explore the mechanism and treatment of human AAA.

Phosphodiesterase 4D contributes to angiotensin II-induced abdominal aortic aneurysm through smooth muscle cell apoptosis

Abdominal aortic aneurysm (AAA) is a permanent expansion of the abdominal aorta that has a high mortality but limited treatment options. Phosphodiesterase (PDE) 4 family members are cAMP-specific hydrolyzing enzymes and have four isoforms (PDE4A-PDE4D). Several pan-PDE4 inhibitors are used clinically. However, the regulation and function of PDE4 in AAA remain largely unknown. Herein, we showed that PDE4D expression is upregulated in human and angiotensin II-induced mouse AAA tissues using RT-PCR, western blotting, and immunohistochemical staining. Furthermore, smooth muscle cell (SMC)-specific Pde4d knockout mice showed significantly reduced vascular destabilization and AAA development in an experimental AAA model. The PDE4 inhibitor rolipram also suppressed vascular pathogenesis and AAA formation in mice. In addition, PDE4D deficiency inhibited caspase 3 cleavage and SMC apoptosis in vivo and in vitro, as shown by bulk RNA-seq, western blotting, flow cytometry and TUNEL staining. Mechanistic studies revealed that PDE4D promotes apoptosis by suppressing the activation of cAMP-activated protein kinase A (PKA) instead of the exchange protein directly activated by cAMP (Epac). Additionally, the phosphorylation of BCL2-antagonist of cell death (Bad) was reversed by PDE4D siRNA in vitro, which indicates that PDE4D regulates SMC apoptosis via the cAMP-PKA-pBad axis. Overall, these findings indicate that PDE4D upregulation in SMCs plays a causative role in AAA development and suggest that pharmacological inhibition of PDE4 may represent a potential therapeutic strategy.

Mouse Abdominal Aortic Aneurysm Model Induced by Perivascular Application of Elastase.

Abdominal aortic aneurysm (AAA), although primarily asymptomatic, is potentially life-threatening as the rupture of AAA usually has a devastating outcome. Currently, there are several distinct experimental models of AAA, each emphasizing a different aspect in the pathogenesis of AAA. The elastase-induced AAA model is the second most used rodent AAA model. This model involves direct infusion or application of porcine pancreatic elastase (PPE) to the infrarenal segment of the aorta. Due to technical challenges, most elastase-induced AAA model nowadays is performed with the external application rather than an intraluminal infusion of PPE. The infiltration of elastase will cause degradation of elastic lamellae in the medial layers, resulting in the loss of aortic wall integrity and subsequent dilation of the abdominal aorta. However, one disadvantage of the elastase-induced AAA model is the inevitable variation of how the surgery is performed. Specifically, the surgical technique of isolating the infrarenal segment of the aorta, the material used for aorta wrapping and PPE incubation, the enzymatic activity of PPE, and the time duration of PPE application can all be important determinants that affect the eventual AAA formation rate and aneurysm diameter. Notably, the difference in these factors from different studies on AAA can lead to reproducibility issues. This article describes a detailed surgical process of the elastase-induced AAA model through direct application of PPE to the adventitia of the infrarenal abdominal aorta in the mouse. Following this procedure, a stable AAA formation rate of around 80% in male and female mice is achievable. The consistency and reproducibility of AAA studies using an elastase-induced AAA model can be significantly enhanced by establishing a standard surgical procedure.

Deciphering Cell-Cell Communication in Abdominal Aortic Aneurysm From Single-Cell RNA Transcriptomic Data.

Cell-cell communication coordinates cellular differentiation, tissue homeostasis, and immune responses in states of health and disease. In abdominal aortic aneurysm (AAA), a relatively common and potentially life-threatening vascular disease, intercellular communications between multiple cell types are not fully understood. In this study, we analyzed published single-cell RNA sequencing (scRNA-seq) datasets generated from the murine CaCl2 model, perivascular elastase model, Angiotensin II model, and human AAA using bioinformatic approaches. We inferred the intercellular communication network in each experimental AAA model and human AAA and predicted commonly altered signaling pathways, paying particular attention to thrombospondin (THBS) signaling between different cell populations. Together, our analysis inferred intercellular signaling in AAA based on single-cell transcriptomics. This work provides important insight into cell-cell communications in AAA and has laid the groundwork for future experimental investigations that can elucidate the cell signaling pathways driving AAA.

The Association Between Curvature and Rupture in a Murine Model of Abdominal Aortic Aneurysm and Dissection.

Mouse models of abdominal aortic aneurysm (AAA) and dissection have proven to be invaluable in the advancement of diagnostics and therapeutics by providing a platform to decipher response variables that are elusive in human populations. One such model involves systemic Angiotensin II (Ang-II) infusion into low density-lipoprotein receptor-deficient (LDLr-/-) mice leading to intramural thrombus formation, inflammation, matrix degradation, dilation, and dissection. Despite its effectiveness, considerable experimental variability has been observed in AAAs taken from our Ang-II infused LDLr-/- mice (n=12) with obvious dissection occurring in 3 samples, outer bulge radii ranging from 0.73 to 2.12 mm, burst pressures ranging from 155 to 540 mmHg, and rupture location occurring 0.05 to 2.53 mm from the peak bulge location. We hypothesized that surface curvature, a fundamental measure of shape, could serve as a useful predictor of AAA failure at supra-physiological inflation pressures. To test this hypothesis, we fit well-known biquadratic surface patches to 360° micro-mechanical test data and used Spearman's rank correlation (rho) to identify relationships between failure metrics and curvature indices. We found the strongest associations between burst pressure and the maximum value of the first principal curvature (rho=-0.591, p-val=0.061), the maximum value of Mean curvature (rho=-0.545, p-val=0.087), and local values of Mean curvature at the burst location (rho=-0.864, p-val=0.001) with only the latter significant after Bonferroni correction. Additionally, the surface profile at failure was predominantly convex and hyperbolic (saddle-shaped) as indicated by a negative sign in the Gaussian curvature. Findings reiterate the importance of shape in experimental models of AAA.

Modulating Neuro-Immune-Induced Macrophage Polarization With Topiramate Attenuates Experimental Abdominal Aortic Aneurysm

The development of abdominal aortic aneurysm (AAA) is attributed to psychological and physical factors. Topiramate, which is an agonist of the GABAA receptor, makes contributions to neuronal disease and is partially involved in immune regulation, may be effective upon abdominal aortic aneurysm progression. We used experimental abdominal aortic aneurysm models: Angiotensin II (Ang II)–induced ApoE−/− male mice (Ang II/APOE model) in our study. In the Ang II/APOE model, all mice (n=64) were divided into four groups: sham group (PBS treatment), control group (Ang II treatment), low-dose group (Ang II + low-dose topiramate, 3 mg/day per mouse), and high-dose group (Ang II + high-dose topiramate, 6 mg/day per mouse). All treatments began on the day after surgery. Moreover, collected tissues and cultured cell were used for histology and biochemical examination. In vitro, the effects of topiramate on bone marrow-derived macrophage stimulated by LPS were investigated. Our data implied that topiramate treatment significantly promoted macrophages preservation and conversion of M1 to M2 macrophage phenotypes in vivo and in vitro. Accordingly, proinflammatory activities mediated by the M1 macrophages were decreased and the repair process mediated by M2 macrophages was enhanced. The low-dose and high-dose groups had abdominal aortic aneurysm incidences of 50% and 37.5%, respectively, compared with 75% in the control group. Topiramate, a promising drug for the psychological disease, that target neuro-immune-induced macrophage polarization may attenuate experimental abdominal aortic aneurysm progression.

SR1001 confers protection against the experimental abdominal aortic aneurysm induced by angiotensin II in ApoE-/-mice

Objective To investigate the effect and mechanism of SR1001 treatment in angiotensin Ⅱ (AngⅡ) induced experimental abdominal aortic aneurysm (EAAA) in apolipoprotein E (ApoE-/-) mice. Methods Forty-five male apolipoprotein E-deficient (ApoE-/-) mice were randomly divided into the following 3 groups: Sham group, AngⅡ model group (Control group), AngⅡ+ SR1001 treatment group (SR1001 group). All mice were treatment with 0.5% dimethylsurfoxide (DMSO) or SR1001 by intraperitoneal injection. The aortic diameter was measured via ultrasound imaging, and the suprarenal aorta was chosen as the measuring point on days 0, 14, 28 thereafter. Twenty-eight days after AngⅡ infusion, the abdominal aortic specimens were harvested. The elastica van Gieson (EVG) staining, and immunofluorescent staining were performed for histopathologic analysis. The protein levels of interleukin (IL)-17, monocyte chemotactic protein (MCP)-1, interferon (IFN)-γ were determined by Western blotting analysis. Results (1) Compared with the Control group, the AAA incidence in the SR1001 group was significantly reduced (33.3% vs. 73.3%, χ2=4.821, P=0.028), Abdominal aorta diameter [(1.39±0.06) mm vs. (1.98±0.11) mm, P=0.009]. However, there was no significant difference in abdominal aorta diameter between the SR1001 group and Sham group [(1.39±0.06) mm vs. (1.07±0.08) mm, P=0.064]. (2) SR1001 treatment in SR1001 group preserves the structure of the aortic wall (P=0.018). (3) SR1001 treatment in SR1001 group attenuates local inflammatory cell infiltration in the EAAA model. (4) SR1001 treatment in SR1001 group suppresses the protein expression of pro-inflammatory mediators (IL-17, MCP-1, IFN-γ) in the EAAA model (P=0.008, 0.016, 0.023, respectively). Conclusion Treatment with SR1001 inhibits the Th17/IL-17A-related inflammatory responses, preserves the structure of the aortic wall, reduces local inflammatory cell infiltration and inflammatory factors expression, and attenuates the progression of abdominal aortic aneurysm. Key words: SR1001; Angiotensin Ⅱ; Apolipoprotein E-/-mice; Interleukin-17; Inflammatory cell; Experimental abdominal aortic aneurysm

Deficiency of γδT cells protects against abdominal aortic aneurysms by regulating phosphoinositide 3-kinase/AKT signaling

ObjectiveIt is known that T lymphocytes are activated in human abdominal aortic aneurysms (AAAs). γδT cells, as a subset of T cells, play a role in many inflammation-related diseases. However, whether γδT cells participate in the formation of AAA remains unknown. In this study, we explored the role of γδT cells in AAA lesions. Methods and ResultsUsing the porcine pancreatic elastase-induced AAA model, we found that knock out of γδT cells significantly attenuated AAA formation. To elucidate how γδT cells contribute to AAA, microarray analysis was performed, which found that the phosphoinositide 3-kinase/AKT signaling pathway was activated in elastase-perfused γδT knockout (γδT KO) mice. By studying differentially expressed genes involved in phosphoinositide 3-kinase signaling, we found that proliferation-related genes (Sos1, Mtor, Myc) were upregulated whereas apoptosis-related genes (Pten, Bcl1, Bad) were downregulated in elastase-perfused γδT KO mice. Furthermore, histopathologic analysis showed increased PCNA+ and decreased TUNEL+ cells in elastase-perfused γδT KO mice compared with wild-type mice. In addition, inflammatory cytokines including interleukin-1β, Mcp-1, and tumor necrosis factor-α were downregulated in the aneurysm tissues of elastase-perfused γδT KO mice. ConclusionsThese data reveal a pathogenic role of γδT cells in the experimental AAA model, likely through mechanisms regulating cell proliferation and mediating inflammatory response. Thus, targeting of γδT cells may offer a potential therapeutic method for aortic aneurysms.

MicroRNA-145 Mediates the Formation of Angiotensin II-Induced Murine Abdominal Aortic Aneurysm.

MicroRNA-145 (miR-145) has been implicated in vascular smooth muscle cell differentiation, but the underlying mechanisms have not been fully understood, especially their role in abdominal aortic aneurysm (AAA) expansion. Here, we sought to explore and define the mechanisms of miR-145 function in the experimental AAA models in AngII-infused ApoE-/- mice. miR-145 was overexpressed in ApoE-/- mice via lentivirus infection, and then the incidence of AAA, maximum abdominal aortic diameter, elastin degradation and MMP2 activation were determined in AngII-infused ApoE-/- mice. In vivo overexpression of miR-145 by lentivirus infection greatly decreased the incidence of AAA, maximum abdominal aortic diameter, and elastin degradation, accompanied with downregulation of MMP2 activation in AngII-infused ApoE-/- mice. Cell culture assays indicated that miR-145 inhibited AngII-induced upregulation of MMP2 gene expression. In contrast, deficiency of MMP2 abolished the effects of miR-145 on AngII-induced elastin and collagens degradations in ApoE-/- mice. These data suggest that regulation of expression of miR-145 may be a potential therapeutic option for vascular disease progression such as AAA expansion.

Cold-inducible RNA-binding protein plays a central role in the pathogenesis of abdominal aortic aneurysm in a murine experimental model

Cold-inducible RNA-binding protein (CIRP) is a recently identified proinflammatory cytokine. We hypothesize that CIRP is involved in the progression of abdominal aortic aneurysms (AAA) and that anti-CIRP treatment could inhibit this progression. We investigated CIRP expression in the sera and aneurysmal tissues of human AAA patients and elastase-induced AAA rats. To further examine the role of CIRP in the development of AAA, anti-CIRP antibody (1mg/kg) or nonimmunized control immunoglobulin (Ig)G (1mg/kg) was injected via the caudal vein in the experimental AAA model. To further investigate the underlying mechanisms, RAW 267.4cells were stimulated with recombinant murine CIRP (rmCIRP). In human AAA tissue, CIRP exhibited a 5.6-fold and 93% increase in mRNA and protein expression, respectively. In a rat AAA model, CIRP was upregulated significantly in a time-dependent manner in the serum and AAA tissue. The anti-CIRP antibody treatment significantly suppressed the dilation of experimental AAA. Simultaneously, inhibition of CIRP significantly attenuated the expression of matrix metalloproteinase (MMP)-2, MMP-9, tumor necrosis factor-α, and monocyte chemoattractant protein-1, and the number of CD68-positive macrophages in the experimental AAA tissue. Invitro, rmCIRP significantly increased MMP-9 messenger RNA expression in a dose-dependent manner by 1.2-fold, 2.9-fold, and 5.5-fold, respectively. Simultaneously, rmCIRP promoted RAW 264.7cell migration, with an approximately 2.7-fold increase in the number of migrated cells. Our findings demonstrate that CIRP mediates experimental AAA development by promoting the inflammatory response and inducing MMP-9 expression, demonstrating its potential as a novel target for inhibiting the progression of AAA.

Myeloid Thrombomodulin Regulates Vascular Inflammation in Abdominal Aortic Aneurysm

Thrombomodulin (TM) is originally described as a marker of endothelium and functions as an anti-coagulant. Apart from its anticoagulant properties, we have recently reported that monocytic TM participated in pathogen-induced inflammatory responses in experimental sepsis models. However, the contribution of TM to vascular inflammation in the development of cardiovascular diseases, such as abdominal aortic aneurysm (AAA), is still poorly understood. CaCl2-induced and angiotensin II-infused experimental models of AAA were used to investigate the role of TM. Myeloid TM-deficient mice (LysMcre/TMflox/flox), not vascular smooth muscle cell TM-deficient mice (SM22-cretg/TMflox/flox mice) exhibited significantly reduced levels of aneurysmal expansion, infiltrating macrophages, proinflammatory cytokines and matrix metalloproteinase-9. Similar results were observed in the ApoE knock-out TM-deficient mice (ApoE-/-/LysMcre/TMflox/flox mice) with infusion of angiotensin II. Moreover, thioglycollate-induced TM-deficient macrophages displayed reduced proinflammatory cytokine profiles, oxidative stress, and adhesion to endothelial cells compared with wild-type mice. Consistent with our observation in the CaCl2-treated murine aortas, we also demonstrated that TM was predominantly located in infiltrating macrophages in human AAA samples. Macrophage infiltration plays an important role in aortic aneurysm, whereas TM expression is involved in the inflammatory responses in macrophages. Decrease of TM expression in macrophages may suppress inflammatory response in vitro as well as aortic aneurysm. These results suggest that targeting to TM-mediated inflammatory responses in monocytes/macrophages could be a new therapeutic target for AAA.

Inhibiting the Th17/IL-17A-related inflammatory responses with digoxin confers protection against experimental abdominal aortic aneurysm.

T helper 17 cells and interleukin-17A have been implicated in the progression of abdominal aortic aneurysm (AAA). Retinoic acid-related orphan receptor gamma thymus, the master transcription factor of T helper 17 cell differentiation, is selectively antagonized by digoxin. However, the effect of antagonizing retinoic acid-related orphan receptor gamma thymus on AAA has not been investigated. We used human aortic sample analysis and 2 different experimental AAA models: (a) Angiotensin II (Ang II)-induced ApoE(-/-) male mice (Ang II/APOE model) and (b) porcine pancreatic elastase perfusion C57BL/6 mice (porcine pancreatic elastase/C57 model). In the Ang II/APOE model, all mice (n=80) were divided into 4 groups: sham group (saline+0.5% dimethyl sulfoxide treatment), control group (Ang II+0.5% dimethyl sulfoxide treatment), low-dose group (Ang II+low-dose digoxin, 20 μg/d per mouse), and high-dose group (Ang II+high-dose digoxin, 40 μg/d per mouse). All treatments began on day 0 after surgery. Efficacy was determined via aortic diameter and systolic blood pressure measurements, histopathology and protein expression, and flow cytometry analysis when euthenized. Human aortic tissue analysis showed that both interleukin-17A and retinoic acid-related orphan receptor gamma thymus increased in AAA tissues. The low-dose and high-dose groups had AAA incidences of 60% and 35%, respectively, compared with 70% in the control group. The T helper 17- and interleukin-17A-related inflammatory responses were dose-dependently attenuated by digoxin treatment. Digoxin was also highly effective in the porcine pancreatic elastase/C57 model. Digoxin attenuates experimental AAA progression in a model-independent manner. Antagonizing retinoic acid-related orphan receptor gamma thymus activity by digoxin may become a novel strategy for nonsurgical AAA treatment.

B2 Cells Suppress Experimental Abdominal Aortic Aneurysms

Recent reports of rupture in patients with abdominal aortic aneurysm (AAA) receiving B-cell depletion therapy highlight the importance of understanding the role of B cells (B1 and B2 subsets) in the development of AAA. We hypothesized that B2 cells aggravate experimental aneurysm formation. The IHC staining revealed infiltration of B cells in the aorta of wild-type (C57BL/6) mice at day 7 after elastase perfusion and persisted through day 21. Quantification of immune cell types using flow cytometry at day 14 showed significantly greater infiltration of mononuclear cells, including B cells (B2: 93% of total B cells) and T cells in elastase-perfused aortas compared with saline-perfused or normal aortas. muMT (mature B-cell deficient) mice were prone to AAA formation similar to wild-type mice in two different experimental AAA models. Contradicting our hypothesis, adoptive transfer of B2 cells suppressed AAA formation (102.0% ± 7.3% versus 75.2% ± 5.5%; P<0.05) with concomitant increase in the splenic regulatory T cell (0.24% ± 0.03% versus 0.92% ± 0.23%; P<0.05) and decrease in aortic infiltration of mononuclear cells. Our data suggest that B2 cells constitute the largest population of B cells in experimental AAA. Furthermore, B2 cells, in the absence of other B-cell subsets, increase splenic regulatory T-cell population and suppress AAA formation.

Abstract 113: Screening of MicroRNAs Expressed in Isolated Cells of Human Abdominal Aortic Aneurysm for the Identification of Potential Biomarkers

Abdominal aortic aneurysm (AAA) is a vascular asymptomatic disease that is one of the leading causes of death in developed countries. Identifying biomarkers for AAA that can be detected easily in blood, before rupture could be useful. The aim of this study was to investigate the potentiality of miRNAS as biomarkers of AAA by performing a profiling of miRNAs expressed in major cells present in the human AAA tissue. The inflammatory cells as macrophages M1 and M2 and smooth muscle cells (SMC) were located by immunohistochemistry in 20 human AAA biopsies, showing a specific distribution towards the aneurysmal aortic wall. The cells were isolated by laser microdissection (LMD) from 20 human AAA biopsies obtained during surgical repair and control SMC from 14 healthy aortic biopsies harvested during organs multiretrieval. RNA extracted from 2 samples of each LMD isolated cells was screened on human miRNAs microarray. MiRNAs were selected with at least a 2-fold change and a detection value threshold corresponding to the value of miR-29b, described in experimental AAA models. Out of the 850 human miRNAs tested for each sample, 408 were found to be present in AAA. Thirty miRNAs were common to each tested cells. Fifty-three miRNAs were found in SMC, of which 12 were specific to AAA compared to control aortas; 86 miRNAs were found in macrophages, of which 11 were specific to M1 macrophages, 37 to M2 macrophages and 38 common to both subtypes. Ten miRNAs were selected to be validated by quantitative RT-PCR in LMD isolated healthy SMC and aneurysmal SMC, M1 and M2 macrophages. We validated 4 miRNAs to be overexpressed in M1 macrophages and 1 overexpressed in M2 macrophages. Two miRs were validated to be less expressed in aneurysmal SMC compared to SMC from normal aortas. MiR-29b expression was specifically down-regulated in aneurysaml SMC compared to normal SMC. In conclusion, the analysis of isolated cells allows to discriminate the miRNAs specifically expressed in inflammatory and vascular cells in AAA and to determine other miRNAs than those described in experimental AAA models as potential biomarkers of AAA.

Abstract 110: B2 Cells Suppress Abdominal Aortic Aneurysms and Increase Peripheral Regulatory T Cells

Objective: Recent reports have documented abdominal aortic aneurysm (AA) rupture in patients receiving B cell depletion therapy. This highlights the importance of understanding the role of B cells (B1 and B2 subtypes) in the development of AA. We hypothesized that B2 cells are abundant in AA and aggravate experimental aneurysm formation. Methods: AAs were induced in WT (C57BL/6) mice by elastase perfusion of the abdominal aorta and harvested at 14 days. Aortic B1 and B2 cells were quantified, and, intracellular cytokine synthesis was determined using flow cytometry. Next, AAs were induced in B cell deficient (muMT) mice using either elastase perfusion or topical elastase application to determine the impact of loss of B cells in AA. Finally, adoptive transfer experiments were performed on muMT mice by tail vein injection of 25x10^6 isolated splenic B2 cells or saline. Regulatory T cell (Treg) population was identified as CD4+ T cell expressing Foxp3 (forkhead box P3). Results: B cells appeared in abdominal aortas at day 7 after elastase perfusion in WT mice and persisted at least till day 21. Compared to saline perfused or normal aortas, elastase perfused aortas had greater infiltration of mononuclear cells including B cells (3631±578 in elastase vs 1476±524 in saline, p=0.02) and T cells. B2 cells constituted 93% of total B cells in AA. Furthermore, B cells isolated from elastase and saline perfused aortas displayed high, but similar levels of intracellular IL-6 and IL-10 expression. Interestingly, in both experimental models of AA, muMT mice were prone to AA formation similar to WT mice as evidenced by elastin degradation or infiltration of T cells, neutrophils and macrophages. Contradicting our hypothesis, adoptive transfer of B2 cells suppressed AA formation (102.0±7.3% vs 75.2±5.5%, p=0.01) with concomitant increase in Treg cells in spleen (0.24±0.03% vs 0.92± 0.23%, p=0.02) and aorta (0.37±0.09% vs 1.09±0.38%, p=0.08), and decrease in aortic infiltration of mononuclear cells. Conclusions: Our data suggest that B2 cells constitute the largest population of B cells in experimental AA and participate in protecting aorta from aneurysm formation at least in part by increasing the Treg cell population.

Effects of Intraluminal Thrombus on Patient-Specific Abdominal Aortic Aneurysm Hemodynamics via Stereoscopic Particle Image Velocity and Computational Fluid Dynamics Modeling

The pathology of the human abdominal aortic aneurysm (AAA) and its relationship to the later complication of intraluminal thrombus (ILT) formation remains unclear. The hemodynamics in the diseased abdominal aorta are hypothesized to be a key contributor to the formation and growth of ILT. The objective of this investigation is to establish a reliable 3D flow visualization method with corresponding validation tests with high confidence in order to provide insight into the basic hemodynamic features for a better understanding of hemodynamics in AAA pathology and seek potential treatment for AAA diseases. A stereoscopic particle image velocity (PIV) experiment was conducted using transparent patient-specific experimental AAA models (with and without ILT) at three axial planes. Results show that before ILT formation, a 3D vortex was generated in the AAA phantom. This geometry-related vortex was not observed after the formation of ILT, indicating its possible role in the subsequent appearance of ILT in this patient. It may indicate that a longer residence time of recirculated blood flow in the aortic lumen due to this vortex caused sufficient shear-induced platelet activation to develop ILT and maintain uniform flow conditions. Additionally, two computational fluid dynamics (CFD) modeling codes (Fluent and an in-house cardiovascular CFD code) were compared with the two-dimensional, three-component velocity stereoscopic PIV data. Results showed that correlation coefficients of the out-of-plane velocity data between PIV and both CFD methods are greater than 0.85, demonstrating good quantitative agreement. The stereoscopic PIV study can be utilized as test case templates for ongoing efforts in cardiovascular CFD solver development. Likewise, it is envisaged that the patient-specific data may provide a benchmark for further studying hemodynamics of actual AAA, ILT, and their convolution effects under physiological conditions for clinical applications.

Inhibition of AAA in a rat model by treatment with ACEI perindopril

BackgroundThe purpose of the present study was to evaluate the effect of a new angiotensin converting enzyme inhibitor perindopril on the formation of experimental abdominal aortic aneurysms (AAAs) in a rat model induced by intraluminal elastase infusion and extraluminal calcium chloride (CaCl2) application. Materials and methodsThirty-six male Sprague–Dawley rats were randomly distributed into three groups (n = 12 per group): model (A), sham (B), and perindopril (C). Rats in model and perindopril groups underwent intra-aortic elastase perfusion and extraluminal CaCl2 application to induce AAAs. Rats in the sham group received aortic perfusion and extraluminal application of saline. A dose of 3 mg/kg/d of perindopril was fed orally in the perindopril group. The maximum abdominal aortic diameter was measured in vivo on days 0 and 28 and by ultrasound on days 7, 14, and 21. The arterial blood pressure was measured directly using a pressure transducer after cannulation in surgery and before death. AAA tissue samples were harvested at day 28 and evaluated using normal hematoxylin and eosin stain, Verhoeff-van Gieson stain for elastin, and image analysis technique. ResultsAortic diameters of rats in the model group consistently increased within 28 d, coinciding with the development of a transmural inflammatory response, thickening of intima, and destruction of the elastic media. Without alteration in blood pressure, the AAA formation rate and mean maximal diameter of aorta at day 28 were significantly lower in the perindopril group compared with the control group (1.71 ± 0.20 versus 2.70 ± 0.69 mm, P < 0.001; 0% versus 90.91%, P < 0.001) and were similar to those in the sham group (1.79 ± 0.29 mm, P = 0.175; 0%, P = 1). The thickness of intima in the perindopril group was lower than that in the model group (20.68 ± 9.96 versus 58.49 ± 32.01 μm, P = 0.001), but higher than that in the sham group (7.23 ± 2.68 μm, P = 0.005). The intensity of elastin fiber showed the opposite trend (0.8541 ± 0.0495 in sham group versus 0.7376 ± 0.1024 in perindopril group versus 0.5413 ± 0.0912 in model group, P < 0.001). ConclusionsPerindopril inhibited the aortic degeneration and AAA formation in the experimental AAA model induced by elastase and CaCl2. This effect, which was independent of its influence on hemodynamics, appeared to be induced by the suppression of the inflammatory cell influx and intimal thickening and the preservation of aortic medial elastin.

Controlled release of ascorbic acid from gelatin hydrogel attenuates abdominal aortic aneurysm formation in rat experimental abdominal aortic aneurysm model

Abdominal aortic aneurysms (AAAs) are associated with oxidative stress and inflammatory response. We investigated the hypothesis that the known antioxidant ascorbic acid, which can also promote elastin and collagen production by smooth muscle cells, would prevent AAA formation in a rat model. An intraluminal elastase and extraluminal calcium chloride-induced rat AAA model was used, and the animals were divided into three groups: control (group C, n = 18), the aorta wrapped with a saline-impregnated gelatin hydrogel sheet (group G, n = 18), and the aorta wrapped with a gelatin hydrogel sheet incorporating ascorbic acid (group A, n = 18). Wrapping of the sheet was completed at the end of treatment for AAA creation. The aortic dilatation ratio was measured, and aortic tissues were further examined for oxidative stress and oxidative DNA damage using biochemical and histologic techniques. Aortic dilatation at both 4 and 8 weeks was inhibited in group A (dilatation ratio [%] at 4 weeks: 186.2 ± 21.8 in group C, 152.3 ± 10.2 in group G, 126.8 ± 11.6 in group A; P < .0001; dilatation ratio [%] at 8 weeks: 219.3 ± 37.5 in group C, 194.0 ± 11.6 in group G, 145.7 ± 8.3 in group A; P = .0002). Elastin and collagen content were significantly preserved in group A (elastin, P = .0015; collagen, P < .0001). The messenger RNA expressions of matrix metalloproteinase (MMP)-9, monocyte chemotactic protein-1, interleukin-1β, and tissue necrosis factor-α (P = .0024, P < .0001, P < .0001, and P < .0001, respectively) were downregulated in group A (P = .0024), whereas tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 were both upregulated in group A (TIMP-1, P = .0014; TIMP-2, P < .0001). Gelatin zymography showed activities of pro-MMP-2, MMP-2, and MMP-9 were significantly suppressed in group C (P < .0001 for each). Reactive oxygen species expression and 8-hydroxydeoxyguanosine and cluster of differentiation 68 staining were significantly suppressed in group A (reactive oxygen species expression, P < .0001; 8-hydroxydeoxyguanosine-positive cells, P < .0001; cluster of differentiation 68 positive cells, P < .0001). Controlled release of ascorbic acid using gelatin hydrogel sheet-attenuated AAA formation through antioxidant and anti-inflammatory effect, regulation of MMP-2, TIMP-1, and TIMP-2, and preserving elastin and collagen in this animal model.

Genetic and Pharmacologic Disruption of Interleukin-1β Signaling Inhibits Experimental Aortic Aneurysm Formation

Abdominal aortic aneurysms (AAAs) are common, but their exact pathogenesis remains unknown and no specific medical therapies are available. We sought to evaluate interleukin-1β (IL-1β) and interleukin-1 receptor (IL-1R) in an experimental AAA model to identify novel therapeutic targets for AAA treatment. IL-1β mRNA and protein levels were significantly elevated in abdominal aortas of 8- to 12-week-old male C57Bl/6 mice after elastase aortic perfusion (wild-type [WT]) compared with saline perfusion. Mice with genetic deletion of IL-1β (IL-1β knockout [KO]) or IL-1R (IL-1R KO) that underwent elastase perfusion demonstrated significant protection against AAA formation, with maximal aortic dilations of 38.0±5.5% for IL-1β KO and 52.5±4.6% for IL-1R KO, compared with 89.4±4.0% for WT mice (P<0.005). Correspondingly, IL-1β KO and IL-1R KO aortas had reduced macrophage and neutrophil staining with greater elastin preservation compared with WT. In WT mice pretreated with escalating doses of the IL-1R antagonist anakinra, there was a dose-dependent decrease in maximal aortic dilation (R=-0.676; P<0.0005). Increasing anakinra doses correlated with decreasing macrophage staining and elastin fragmentation. Lastly, WT mice treated with anakinra 3 or 7 days after AAA initiation with elastase demonstrated significant protection against AAA progression and had decreased aortic dilation compared with control mice. IL-1β is critical for AAA initiation and progression, and IL-1β neutralization through genetic deletion or receptor antagonism attenuates experimental AAA formation. Disrupting IL-1β signaling offers a novel pathway for AAA treatment.