Endothelial pannexin-1 channels modulate macrophage and smooth muscle cell activation in abdominal aortic aneurysm formation.

Pannexin-1 (Panx1) channels have been shown to regulate leukocyte trafficking and tissue inflammation but the mechanism of Panx1 in chronic vascular diseases like abdominal aortic aneurysms (AAA) is unknown. Here we demonstrate that Panx1 on endothelial cells, but not smooth muscle cells, orchestrate a cascade of signaling events to mediate vascular inflammation and remodeling. Mechanistically, Panx1 on endothelial cells acts as a conduit for ATP release that stimulates macrophage activation via P2X7 receptors and mitochondrial DNA release to increase IL-1ß and HMGB1 secretion. Secondly, Panx1 signaling regulates smooth muscle cell-dependent intracellular Ca2+ release and vascular remodeling via P2Y2 receptors. Panx1 blockade using probenecid markedly inhibits leukocyte transmigration, aortic inflammation and remodeling to mitigate AAA formation. Panx1 expression is upregulated in human AAAs and retrospective clinical data demonstrated reduced mortality in aortic aneurysm patients treated with Panx1 inhibitors. Collectively, these data identify Panx1 signaling as a contributory mechanism of AAA formation.

Tightrope Technique for facilitating complex endovascular aortic repair in patients with severely angulated neck.

An 84-year-old presented with a large, symptomatic juxtarenal abdominal aortic aneurysm. Owing to severe angulation of the infrarenal neck, advancement of the distal bifurcated component caused dramatic lateral movement of the proximal physician-modified endovascular graft (PMEG) fenestrated device. This procedure risked aneurysm sac perforation and possible PMEG device displacement. To avoid this complication, the distal aspect of the PMEG device was tethered in place using endoscopic forceps to provide countertraction, similar to pulling a tightrope. This technique allowed for the uneventful placement of the distal bifurcated component without complication. This technique can overcome device placement challenges within an angulated aorta caused by large aneurysms.

B Cell-Activating Factor Antagonism Attenuates the Growth of Experimental Abdominal Aortic Aneurysm.

B cell-activating factor (BAFF), part of a tumor necrosis factor family of cytokines, was recently identified as a regulator of atherosclerosis; however, its role in aortic aneurysm has not been determined. Here, the study examined the effect of selective BAFF antagonism using an anti-BAFF antibody (blocks binding of BAFF to receptors BAFF receptor 3, transmembrane activator and CAML interactor, and B-cell maturation antigen) and mBaffR-mFc (blocks binding of BAFF to BAFF receptor 3) on a murine model of abdominal aortic aneurysm (AAA). In a prevention strategy, the antagonists were injected before the induction of AAA, and in an intervention strategy, the antagonists were injected after the induction of AAA. Both strategies attenuated the formation of AAA. In the intervention group, BAFF antagonism depleted most of the mature B-cell subsets in spleen and circulation, leading to enhanced resolution of inflammation in AAA as indicated by decreased infiltration of B cells and proinflammatory macrophages and a reduced number of apoptotic cells. In AAA tissues, B cells and macrophages were found in close contact. In vitro, B cells, irrespective of treatment with BAFF, impaired the efferocytosis activity of macrophages, suggesting a direct innate role of B cells on macrophage function. Altogether, BAFF antagonism affects survival of the mature B cells, promotes resolution of inflammation in the aorta, and attenuates the growth of AAA in mice.

Sex-Based Differences Among Experimental Swine Abdominal Aortic aneurysms.

BACKGROUND: Female sex protects against abdominal aortic aneurysms (AAAs); however, the mechanisms behind these sex-based differences remain unknown. The purpose of this study was to explore the role of sex and sex hormones in AAA formation among swine. MATERIALS AND METHODS: Using a previous validated model, infrarenal AAA were surgically created in uncastrated male (n = 8), female (n = 5), and castrated male (n = 4) swine. Aortic dilation was measured on postoperative day 28 during the terminal procedure and compared to initial aortic diameter measured during the index procedure. Tissue was analyzed for immunohistochemistry, cytokine array, gelatin zymography, serum 17ß-estradiol, and testosterone assay. RESULTS: Uncastrated males had significantly larger maximal aortic dilation compared to castrated males (113.5% ± 11.4% versus 38.1% ± 4.5%, P = 0.0012). Females had significantly higher mean aortic dilation compared to castrated males (96.2% ± 7.5% versus 38.1% ± 4.5%, P = 0.0004). Aortic diameters between females and uncastrated males were not significantly different on day 28. Female swine had significantly higher concentrations of 17ß-estradiol compared with uncastrated males (1590 ± 873.3 ng/mL versus 95.2 ± 2.3 ng/mL, P = 0.047), with no significant difference between females and castrated males. Uncastrated male AAA demonstrated significantly more elastin degradation compared with female and castrated males (P = 0.01 and <0 .01, respectively). No differences existed for T-cells or smooth muscle cells between groups. Multiple proinflammatory cytokines were elevated within uncastrated male aortic walls compared to females and castrated males. CONCLUSIONS: Sex hormones, specifically 17ß-estradiol and testosterone, influence experimental swine AAA formation as demonstrated by increased aneurysm size, collagen turnover, and elastolysis in uncastrated males in processes reflective of human disease.

Pharmacologic inhibition of transient receptor channel vanilloid 4 attenuates abdominal aortic aneurysm formation.

Abdominal aortic aneurysm (AAA) formation is characterized by inflammation, leukocyte infiltration, and vascular remodeling. This study investigates the role of TRPV4 channels, which are transmembrane calcium channels that can regulate vascular tone, in modulating AAA formation. The elastase-treatment model of AAA in C57BL6 (WT) mice and Angiotensin II treatment model in ApoE-/- mice were used to confirm our hypotheses. The administration of a specific TRPV4 antagonist, GSK2193874, in elastase-treated WT mice and in AngII-treated ApoE-/- mice caused a significant attenuation of aortic diameter, decrease in pro-inflammatory cytokines (IL-1ß, IL-6, IL-17, MCP-1, MIP-1α, MIP-2, RANTES, and TNF-α), inflammatory cell infiltration (CD3 + T cells, macrophages, and neutrophils), elastic fiber disruption, and an increase in smooth muscle cell α-actin expression compared to untreated mice. Similarly, elastase-treated TRPV4-/- mice had a significant decrease in AAA formation, aortic inflammation, and vascular remodeling compared to elastase-treated WT mice on Day 14. In vitro studies demonstrated that the inhibition of TRPV4 channels mitigates aortic smooth muscle cell-dependent inflammatory cytokine production as well as decreases neutrophil transmigration through aortic endothelial cells. Therefore, our results suggest that TRPV4 antagonism can attenuate aortic inflammation and remodeling via decreased smooth muscle cell activation and neutrophil transendothelial migration during AAA formation.

Single-Photon Emission Computed Tomography Imaging Using Formyl Peptide Receptor 1 Ligand Can Diagnose Aortic Aneurysms in a Mouse Model.

BACKGROUND: Our previous studies showed that neutrophil infiltration and activation plays an important role in the pathogenesis of abdominal aortic aneurysms (AAA). However, there is a lack of noninvasive, inflammatory cell-specific molecular imaging methods to provide early diagnosis of AAA formation. Formyl peptide receptor 1 (FPR1) is rapidly upregulated on neutrophils during inflammation. Therefore, it is hypothesized that the use of cinnamoyl-F-(D)L-F-(D)L-F-K (cFLFLF), a PEGylated peptide ligand that binds FPR1 on activated neutrophils, would permit accurate and noninvasive diagnosis of AAA via single-photon emission computed tomography (SPECT) imaging. MATERIALS AND METHODS: Male C57BL/6 (wild-type) mice were treated with topical elastase (0.4 U/mL type 1 porcine pancreatic elastase) or heat-inactivated elastase (control), and aortic diameter was measured by video micrometry. Comparative histology was performed on Day 14 to assess neutrophil infiltration in aortic tissue. We performed near-infrared fluorescence imaging using c-FLFLF-Cy7 probe on Days 7 and 14 postelastase treatment and measured fluorescence intensity ex vivo in excised aortic tissue. A separate group of animals were injected with 99mTc-c-FLFLF 2 h before SPECT imaging on Day 14 using a SPECT/computed tomography/positron emission tomography trimodal scanner. Coexpression of neutrophils with c-FLFLF was also performed on aortic tissue by immunostaining on Day 14. RESULTS: Aortic diameter was significantly increased in the elastase group compared with controls on Days 7 and 14. Simultaneously, a marked increase in neutrophil infiltration and elastin degradation as well as decrease in smooth muscle integrity were observed in aortic tissue after elastase treatment compared with controls. Moreover, a significant increase in fluorescence intensity of c-FLFLF-Cy7 imaging probe was also observed in elastase-treated mice on Day 7 (approximately twofold increase) and Day 14 (approximately 2.5-fold increase) compared with respective controls. SPECT imaging demonstrated a multifold increase in signal intensity for 99mTc-cFLFLF radiolabel probe in mice with AAA compared with controls on Day 14. Immunostaining of aortic tissue with c-FLFLF-Cy5 demonstrated a marked increase in coexpression with neutrophils in AAA compared with controls. CONCLUSIONS: cFLFLF, a novel FPR1 ligand, enables quantifiable, noninvasive diagnosis and progression of AAAs. Clinical application of this inflammatory, cell-specific molecular probe using SPECT imaging may permit early diagnosis of AAA formation, enabling targeted therapeutic interventions and preventing impending aortic rupture.

Porcine Model of Infrarenal Abdominal Aortic Aneurysm.

Large animal models to study abdominal aortic aneurysms are sparse. The purpose of this model is to create reproducible, clinically significant infrarenal abdominal aortic aneurysms (AAA) in swine. To achieve this, we use a combination of balloon angioplasty, elastase and collagenase, and a lysyl oxidase inhibitor, called ß-aminopropionitrile (BAPN), to create clinically significant infrarenal aortic aneurysms, analogous to human disease. Noncastrated male swine are fed BAPN for 7 days prior to surgery to achieve a steady state in the blood. A midline laparotomy is performed and the infrarenal aorta is circumferentially dissected. An initial measurement is recorded prior to aneurysm induction with a combination of balloon angioplasty, elastase (500 units)/collagenase (8000 units) perfusion, and topical elastase application. Swine are fed BAPN daily until terminal procedure on either postoperative day 7, 14, or 28, at which time the aneurysm is measured, and tissue procured. BAPN + surgery pigs are compared to pigs that underwent surgery alone. Swine treated with BAPN and surgery had a mean aortic dilation of 89.9% ± 47.4% at day 7, 105.4% ± 58.1% at day 14, and 113.5% ± 30.2% at day 28. Pigs treated with surgery alone had significantly smaller aneurysms compared to BAPN + surgery animals at day 28 (p < 0.0003). The BAPN + surgery group had macroscopic and immunohistochemical evidence of end stage aneurysmal disease. Clinically significant infrarenal AAA can be induced using balloon angioplasty, elastase/collagenase perfusion and topical application, supplemented with oral BAPN. This model creates large, clinically significant AAA with hallmarks of human disease. This has important implications for the elucidation of AAA pathogenesis and testing of novel therapies and devices for the treatment of AAA. Limitations of the model include variation in BAPN ingested by swine, quality of elastase perfusion, and cost of BAPN.

A novel swine model of abdominal aortic aneurysm.

OBJECTIVE: Few large-animal models exist for the study of aortic aneurysms. ß-Aminopropionitrile (BAPN) is a compound known to cause aortic aneurysms by inhibiting lysyl oxidase, a collagen cross-linking enzyme. It is hypothesized that BAPN plus aneurysm induction surgery would result in significant aneurysm formation in swine with biologic properties similar to human disease. METHODS: Initial experiments were performed in uncastrated male swine not treated with BAPN (surgery alone). Subsequently, uncastrated male swine were fed BAPN (0.15 g/kg) for 7 days before undergoing surgery; the infrarenal aorta was circumferentially dissected and measured, balloon dilated, and perfused with elastase (500 units) and type I collagenase (8000 units), with extraluminal elastase application. In the BAPN groups, daily BAPN feedings continued until swine harvest at postoperative days 7, 14, and 28. RESULTS: Swine undergoing surgery alone (n = 12) had significantly less dilation at 28 days compared with BAPN + surgery swine (51.9% ± 29.2% [0%-100%] vs 113.5% ± 30.2% [52.9%-146.2%]; P < .0003). Mean aortic dilation in animals undergoing treatment with surgery and BAPN was 86.9% ± 47.4% (range, 55.6%-157.1%), 105.4% ± 58.1% (50%-133.3%), and 113.5% ± 30.2% (52.9%-146.2%) at 7, 14, and 28 days, respectively. In the BAPN + surgery group, significant elastolysis was present at all time points, whereas aortic wall collagen content was not significantly different. Smooth muscle cells were significantly depleted at 14 and 28 days, and M1 macrophages were increased at 14 and 28 days (P < .05, all). Matrix metalloproteinase 2 was elevated at 7 days (P < .05). Multiple proinflammatory cytokines were elevated within the aortic wall of BAPN + surgery swine. CONCLUSIONS: BAPN plus surgery resulted in significantly larger aortic aneurysms than surgery alone and was critical to aneurysm formation in this novel swine model. Hallmarks of human disease, such as elastin fragmentation, smooth muscle cell depletion, macrophage infiltration, matrix metalloproteinase activation, and proinflammatory cytokine expression, were observed in BAPN-treated swine. This model better parallels many of the characteristics of human AAAs and may be suitable for prehuman drug trials.

Tamsulosin attenuates abdominal aortic aneurysm growth.

BACKGROUND: Tamsulosin, an α1A-adrenergic receptor inhibitor, is prescribed to treat benign prostatic hyperplasia in men >60 years of age, the same demographic most susceptible to abdominal aortic aneurysm. The goal of this study was to investigate the effect of tamsulosin on abdominal aortic aneurysm pathogenesis. METHODS: Abdominal aortic aneurysms were induced in WT C57BL/6 male mice (n = 9-18/group), using an established topical elastase abdominal aortic aneurysm model. Osmotic pumps were implanted in mice 5 days before operation to create the model, administering either low dose (0.125 µg/day tamsulosin), high dose (0.250µg/day tamsulosin), or vehicle treatments with and without topical application of elastase. Blood pressures were measured preoperatively and on postoperative days 0, 3, 7, and 14. On postoperative day 14, aortic diameter was measured before harvest. Sample aortas were prepared for histology and cytokine analysis. RESULTS: Measurements of systolic blood pressure did not differ between groups. Mice treated with the low dose of tamsulosin and with the high dose of tamsulosin showed decreased aortic diameter compared with vehicle-treated control (93% ± 24 versus 94% ± 30 versus 132% ± 24, respectively; P = .0003, P = .0003). Cytokine analysis demonstrated downregulation of pro-inflammatory cytokines in both treatment groups compared with the control (P < .05). Histology exhibited preservation of elastin in both low- and high-dose tamsulosin-treated groups (P = .0041 and P = .0018, respectively). CONCLUSION: Tamsulosin attenuates abdominal aortic aneurysm formation with increased preservation of elastin and decreased production of pro-inflammatory cytokines. Further studies are necessary to elucidate the mechanism by which tamsulosin attenuates abdominal aortic aneurysm pathogenesis.

A novel reproducible model of aortic aneurysm rupture.

INTRODUCTION: Given the unknown biologic antecedents before aortic aneurysm rupture, the purpose of this study was to establish a reproducible model of aortic aneurysm rupture. METHODS: We fed 7-week-old apolipoprotein E deficient mice a high-fat diet for 4 weeks and osmotic infusion pumps containing Angiotensin II were implanted. Angiotensin II was delivered continuously for 4 weeks at either 1,000 ng/kg/min (n = 25) or 2,000 ng/kg/min (n = 29). A third group (n = 14) were given Angiotensin II at 2,000 ng/kg/min and 0.2% ß-aminopropionitrile dissolved in drinking water. Surviving mice were killed 28 days after pump placement, aortic diameters were measured, and molecular analyses were performed. RESULTS: Survival at 28 days was significantly different among groups with 80% survival in the 1,000 ng/kg/min group, 52% in the 2,000 ng/kg/min group, and only 14% in the Angiotensin II/ß-aminopropionitrile group (P = .0001). Concordantly, rupture rates were statistically different among groups (8% versus 38% versus 79%, P < .0001). Rates of abdominal aortic aneurysm were 48%, 55%, and 93%, respectively, with statistically higher rates in the Angiotensin II/ß-aminopropionitrile group compared with both the 1,000 ng and 2,000 ng Angiotensin II groups (P = .006 and P = .0165, respectively). Rates of thoracic aortic aneurysm formation were 12%, 52%, and 79% in the 3 groups with a statistically higher rate in the Angiotensin II/ß-aminopropionitrile group compared with 1,000 ng group (P < .0001). CONCLUSIONS: A reproducible model of aortic aneurysm rupture was developed with a high incidence of abdominal and thoracic aortic aneurysm. This model should enable further studies investigating the pathogenesis of aortic rupture, as well as allow for targeted strategies to prevent human aortic aneurysm rupture.