Porcine Pancreatic Elastase Infusion Research Articles

Abstract 3013: Indoleamine 2,3-dioxygenase-mediated Tryptophan Catabolism Limits Experimental Abdominal Aortic Aneurysms

Background: Indoleamine 2,3-dioxygenase (IDO) promotes immunosuppression. Genetic IDO deficiency limits angiotensin II-induced abdominal aortic aneurysms (AAAs) in hyperlipidemic mice. We investigated the influence of IDO inhibition or IDO-catabolized tryptophan catabolite on elastase-induced AAAs in normolipidemic mice. Methods: AAAs were induced in male C57B/6J mice via intra-aortic porcine pancreatic elastase (PPE) infusion. Mice were treated with IDO inhibitor 1-methyl-DL-tryptophan (MT, 2 mg/ml in drinking water), tryptophan metabolite 3-hydroxyanthranilic acid (HAA, 200 mg/kg, oral gavage), or vehicle, starting 3 days prior to PPE infusion for 17 days. AAAs were assessed via ultrasonography and histopathology at sacrifice. Results: The Figure summarizes aortic diameters and histopathology in differentially treated PPE-infused mice. Rapid aortic enlargement was noted in MT-, as compared to vehicle-treated mice following PPE infusion. AAA rupture, unusual for this model, was noted in MT-treated AAA mice. No difference was noted between groups in medial elastin degradation, smooth muscle cell depletion, or macrophage accumulation. However, mural neoangiogenesis and lymphocyte accumulation were remarkably augmented in MT-treated mice. HAA Treatment attenuated aortic enlargement in conjunction with improved medial elastin and smooth muscle cell retention and reduced aortic leukocyte accumulation and neovessels. Conclusion: IDO activity and its tryptophan metabolite HAA limit experimental AAA progression in the PPE model. These results provide further support for investigating tryptophan metabolites in clinical AAA disease suppression.

C-reactive protein deficiency ameliorates experimental abdominal aortic aneurysms.

C-reactive protein (CRP) levels are elevated in patients with abdominal aortic aneurysms (AAA). However, it has not been investigated whether CRP contributes to AAA pathogenesis. CRP deficient and wild type (WT) male mice were subjected to AAA induction via transient intra-aortic infusion of porcine pancreatic elastase. AAAs were monitored by in situ measurements of maximal infrarenal aortic external diameters immediately prior to and 14 days following elastase infusion. Key AAA pathologies were assessed by histochemical and immunohistochemical staining procedures. The influence of CRP deficiency on macrophage activation was evaluated in peritoneal macrophages in vitro. CRP protein levels were higher in aneurysmal than that in non-aneurysmal aortas. Aneurysmal aortic dilation was markedly suppressed in CRP deficient (aortic diameter: 1.08 ± 0.11mm) as compared to WT (1.21 ± 0.08mm) mice on day 14 after elastase infusion. More medial elastin was retained in CRP deficient than in WT elastase-infused mice. Macrophage accumulation was significantly less in aneurysmal aorta from CRP deficient than that from WT mice. Matrix metalloproteinase 2 expression was also attenuated in CRP deficient as compared to WT aneurysmal aortas. CRP deficiency had no recognizable influence on medial smooth muscle loss, lymphocyte accumulation, aneurysmal angiogenesis, and matrix metalloproteinase 9 expression. In in vitro assays, mRNA levels for tumor necrosis factor α and cyclooxygenase 2 were reduced in lipopolysaccharide activated peritoneal macrophages from CRP deficient as compared to wild type mice. CRP deficiency suppressed experimental AAAs by attenuating aneurysmal elastin destruction, macrophage accumulation and matrix metalloproteinase 2 expression.

Mechanisms and efficacy of metformin-mediated suppression of established experimental abdominal aortic aneurysms

ObjectiveMetformin treatment attenuates experimental abdominal aortic aneurysm (AAA) formation, as well as reduces clinical AAA diameter enlargement in patients with diabetes. The mechanisms of metformin-mediated aneurysm suppression, and its efficacy in suppressing established experimental aneurysms, remain uncertain. MethodsExperimental AAAs were created in male C57BL/6J mice via intra-aortic infusion of porcine pancreatic elastase. Metformin alone (250 mg/kg), or metformin combined with the 5′ AMP-activated protein kinase (AMPK) antagonist Compound C (10 mg/kg), were administered to respective mouse cohorts daily beginning 4 days following AAA induction. Further AAA cohorts received either the AMPK agonist AICA riboside (500 mg/kg) as positive, or vehicle (saline) as negative, controls. AAA progression in all groups was assessed via serial in vivo ultrasonography and histopathology at sacrifice. Cytokine-producing T cells and myeloid cellularity were determined by flow cytometric analyses. ResultsMetformin limited established experimental AAA progression at 3 (−85%) and 10 (−68%) days following treatment initiation compared with saline control. Concurrent Compound C treatment reduced this effect by approximately 50%. In metformin-treated mice, reduced AAA progression was associated with relative elastin preservation, smooth muscle cell preservation, and reduced mural leukocyte infiltration and neoangiogenesis compared with vehicle control group. Metformin also resulted in reduced interferon-γ-, but not interleukin-10 or -17, producing splenic T cells in aneurysmal mice. Additionally, metformin therapy increased circulating and splenic inflammatory monocytes (CD11b+Ly-6Chigh), but not neutrophils (CD11b+Ly-6G+), with no effect on respective bone marrow cell populations. ConclusionsMetformin treatment suppresses existing experimental AAA progression in part via AMPK agonist activity, limiting interferon-γ-producing T cell differentiation while enhancing circulating and splenic inflammatory monocyte retention.

Type I Interferon Receptor Subunit 1 Deletion Attenuates Experimental Abdominal Aortic Aneurysm Formation.

Objective: Type I interferon receptor signaling contributes to several autoimmune and vascular diseases such as lupus, atherosclerosis and stroke. The purpose of this study was to assess the influence of type I interferon receptor deficiency on the formation and progression of experimental abdominal aortic aneurysms (AAAs). Methods: AAAs were induced in type I interferon receptor subunit 1 (IFNAR1)-deficient and wild type control male mice via intra-infrarenal aortic infusion of porcine pancreatic elastase. Immunostaining for IFNAR1 was evaluated in experimental and clinical aneurysmal abdominal aortae. The initiation and progression of experimental AAAs were assessed via ultrasound imaging prior to (day 0) and days 3, 7 and 14 following elastase infusion. Aneurysmal histopathology was analyzed at sacrifice. Results: Increased aortic medial and adventitial IFNAR1 expression was present in both clinical AAAs harvested at surgery and experimental AAAs. Following AAA induction, wild type mice experienced progressive, time-dependent infrarenal aortic enlargement. This progression was substantially attenuated in IFNAR1-deficient mice. On histological analyses, medial elastin degradation, smooth muscle cell depletion, leukocyte accumulation and neoangiogenesis were markedly diminished in IFNAR1-deficient mice in comparison to wild type mice. Conclusion: IFNAR1 deficiency limited experimental AAA progression in response to intra-aortic elastase infusion. Combined with clinical observations, these results suggest an important role for IFNAR1 activity in AAA pathogenesis.

Abstract 295: Methyl Transferase Inhibitor 5-azacytidine Suppresses Experimental Abdominal Aortic Aneurysms

Background: Increased vascular DNA methylation is present in disease states including abdominal aortic aneurysms (AAA). The impact of dysregulated DNA methylation on aneurysm pathogenesis, however, remains uncertain. We evaluated the influence of 5-azacytine (AZA), a methyl transferase inhibitor, on experimental AAA initiation and progression. Methods and Results: AAAs were induced in male C57BL/6J mice via intra-aortic porcine pancreatic elastase infusion. AZA or vehicle were administered intraperitoneally starting one day prior to (prophylactic, 2 mg/kg), or 4 days following (therapeutic, 1 or 2 mg/kg), elastase infusion and continuing daily through 14 days. AAA development and progression were evaluated by serial ultrasonographic aortic diameter measurements and histopathology at sacrifice.Time dependent post-elastase aortic diameter enlargement was markedly reduced in mice receiving prophylactic AZA treatment. A substantial delay in AAA onset was noted as well. While therapeutic AZA tended to mitigate further aneurysmal enlargement at either dose, a significant reduction was noted only in the 2 mg/kg group at 14 days. On histopathological assessment, AZA was associated with marked attenuation of characteristic aneurysmal pathologic features including medial elastin and smooth muscle destruction, leukocyte accumulation and neoangiogenesis in all groups. Conclusion: AZA treatment attenuates aneurysm development and progression in the intra-aortic porcine pancreatic elastase infusion AAA model. Pharmacologic strategies effective at limiting or reducing DNA methylation may have translational applications in the medical management of AAA disease.

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.

Dapagliflozin Ameliorates the Formation and Progression of Experimental Abdominal Aortic Aneurysms by Reducing Aortic Inflammation in Mice.

Background Dapagliflozin, a sodium glucose transporter protein-2 (SGLT-2) inhibitor, reduces the risk for cardiovascular diseases. However, the influence of dapagliflozin on nondissecting abdominal aortic aneurysms (AAAs) remains unclear. Methods AAAs were created in male C57BL/6 mice via intra-aortic porcine pancreatic elastase (PPE) infusion. Mice were daily treated with dapagliflozin (1 or 5 mg/kg body weight) or an equal volume of vehicle through oral gavage beginning one day prior to PPE infusion for 14 days. To investigate its translational value, dapagliflozin or vehicle was also administered to mice with existing AAAs in another cohort. Aortic diameters were measured prior to (day 0 for baseline) and 14 days after PPE infusion. After sacrifice, mice aortae were collected, and following histological analyses were performed. Results Dapagliflozin treatment significantly reduced aneurysmal aortic expansion following PPE infusion as compared to vehicle treatment especially at 5 mg/kg body weight (approximately 21% and 33% decreases in 1 and 5 mg/kg treatment groups, respectively). The dose-dependent attenuation of AAAs by dapagliflozin was also confirmed on histological analyses. Dapagliflozin remarkably reduced aortic accumulation of macrophages, CD4+ T cells, and B cells particularly following dapagliflozin treatment at 5 mg/kg. Dapagliflozin treatment also markedly attenuated medial SMC loss. Though the difference was not significant, dapagliflozin treatment tended to attenuate CD8+ T cells and elastin degradation. Dapagliflozin treatment at 5 mg/kg caused a 53% reduction in neovessel density. Furthermore, dapagliflozin treatment mitigated further progress of existing AAAs. Conclusion Dapagliflozin treatment ameliorated PPE-induced AAAs by inhibiting aortic leukocytes infiltration and angiogenesis.

Effects of hypercholesterolism on expansion of abdominal aortic aneurysm in rat model

BackgroundInflammation is recognized as a critical process in expansion of abdominal aortic aneurysm (AAA). A relationship between effects of cholesterol and statin in this process have been suggested, but remain untested. Therefore, current study aimed to examine the effects of hypercholesterolism on expansion of AAA in a rat model.MethodsA total of 16 male rats were divided into 4 groups as follows: group I, normocholesterol diet and saline infusion, group II, normocholesterol diet and porcine pancreatic elastase (PPE) infusion, group III, hypercholesterol diet and PPE infusion, and group IV, hypercholesterol diet, PPE infusion and statin administration. At the 3rd week, saline was infused intraluminally in group I and PPE in groups II-IV to induce AAA. At the 5th week, blood and aortic tissue were obtained from each rat for evaluation of lipid profiles, aortic diameters (ADs), and characteristics of stains.ResultsPost-procedural aortic diameter (AD3) and AD3/pre-procedural aortic diameter (AD1) were significantly different among four groups (P = 0.042, P = 0.028, respectively). AD3 was significantly larger in group II than group I, and group III than group IV (P = 0.012, P = 0.043, respectively). AD3/AD1 was significantly higher in group II than group I, and group III than group II (P = 0.008, P = 0.030, respectively). Group III showed the highest cellularity for inflammatory cells.ConclusionsThough larger experimental and clinical studies are necessary, authors suggest that hypercholesterolism can aggravate expansion of AAA, and that statin therapy can reduce it. Therefore, monitoring for hypercholesterolism and instituting statin therapy may be helpful to suppress expansion of AAA.

No Effect of Hypercholesterolemia on Elastase-Induced Experimental Abdominal Aortic Aneurysm Progression.

Objective: Epidemiological studies link hyperlipidemia with increased risk for abdominal aortic aneurysms (AAAs). However, the influence of lipid-lowering drugs statins on prevalence and progression of clinical and experimental AAAs varies between reports, engendering controversy on the association of hyperlipidemia with AAA disease. This study investigated the impact of hypercholesterolemia on elastase-induced experimental AAAs in mice. Methods: Both spontaneous (targeted deletion of apolipoprotein E) and induced mouse hypercholesterolemia models were employed. In male wild type (WT) C57BL/6J mice, hypercholesterolemia was induced via intraperitoneal injection of an adeno-associated virus (AAV) encoding a gain-of-function proprotein convertase subtilisin/kexin type 9 mutation (PCSK9) followed by the administration of a high-fat diet (HFD) (PCSK9+HFD) for two weeks. As normocholesterolemic controls for PCSK9+HFD mice, WT mice were infected with PCSK9 AAV and fed normal chow, or injected with phosphate-buffered saline alone and fed HFD chow. AAAs were induced in all mice by intra-aortic infusion of porcine pancreatic elastase and assessed by ultrasonography and histopathology. Results: In spontaneous hyper- and normo-cholesterolemic male mice, the aortic diameter enlarged at a constant rate from day 3 through day 14 following elastase infusion. AAAs, defined as a more than 50% diameter increase over baseline measurements, formed in all mice. AAA progression was more pronounced in male mice, with or without spontaneous hyperlipidemia. The extent of elastin degradation and smooth muscle cell depletion were similar in spontaneous hyper- (score 3.5 for elastin and 4.0 for smooth muscle) and normo- (both scores 4.0) cholesterolemic male mice. Aortic mural macrophage accumulation was also equivalent between the two groups. No differences were observed in aortic accumulation of CD4+ or CD8+ T cells, B cells, or mural angiogenesis between male spontaneous hyper- and normocholesterolemic mice. Similarly, no influence of spontaneous hypercholesterolemia on characteristic aneurysmal histopathology was noted in female mice. In confirmatory experiments, induced hypercholesterolemia also exerted no appreciable effect on AAA progression and histopathologies. Conclusion: This study demonstrated no recognizable impact of hypercholesterolemia on elastase-induced experimental AAA progression in both spontaneous and induced hypercholesterolemia mouse models. These results add further uncertainty to the controversy surrounding the efficacy of statin therapy in clinical AAA disease.

Betanin Prevents Experimental Abdominal Aortic Aneurysm Progression by Modulating the TLR4/NF-κB and Nrf2/HO-1 Pathways.

Betanin, a bioactive ingredient mostly isolated from beetroots, exhibits a protective effect against cardiovascular diseases. However, its effects on abdominal aortic aneurysm (AAA) have not been elucidated. In this study, an AAA model was constructed by infusion of porcine pancreatic elastase in C57BL/6 mice. Mice were then administered with betanin or saline intragastrically once daily for 14 d. Our results showed that treatment with betanin remarkably limited AAA enlargement and mitigated the infiltration of inflammatory cells in the adventitia. The increased expression of proinflammatory cytokines and matrix metalloproteinases (MMPs) was also significantly alleviated following betanin treatment. Furthermore, betanin suppressed the activation of toll-like receptor 4 (TLR4)/nuclear factor-kappaB (NF-κB) signaling in the aortic wall, and downregulated the levels of tissue-reactive oxygen species as well as circulating 8-isoprostane by stimulating the nuclear factor-E2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway. Taken together, these data suggest that betanin may attenuate AAA progression and may be used as a therapeutic drug against AAA.

Paeonol Ameliorates Abdominal Aortic Aneurysm Progression by the NF-κB Pathway

Abdominal aortic aneurysm (AAA) is a chronic inflammatory disease characterized by localized progressive dilatation. Currently, paeonol has been shown to possess anti-inflammatory and protective cardiovascular properties. Our study aimed to investigate the potential influences of paeonol on AAA progression. Experimental AAAs were created in C57BL/6J mice by intra-aortic infusion of porcine pancreatic elastase, and then intragastrically administered paeonol (20 mg/kg/day) for 14 days. The effects of paeonol on experimental AAA were measured by ultrasound imaging, histopathology, and western blot analyses. Paeonol treatment limited the enlargement of the aneurysmal diameter and alleviated the depletion of elastic fibers and vascular smooth muscle cells (VSMCs). Furthermore, the infiltration of CD68+ macrophages and CD8+ lymphocytes was obviously attenuated after paeonol administration, along with mural neoangiogenesis. Western blot results showed that paeonol inhibited the expression of matrix metalloproteinase (MMP) and the NF-κB pathway activation. Paeonol might prevent experimental AAA progression by inhibiting the NF-κB pathway, which suggests that it is a potential drug for AAA.

Inhibition of miR-188-5p Suppresses Progression of Experimental Abdominal Aortic Aneurysms.

Abdominal aortic aneurysm (AAA) is an aging-related degenerative disease. miR-188-5p was reported to induce cell senescence and play a key role in aging-related disease. Therefore, in this study, we investigated miR-188-5p expression during progression in experimental AAAs. Furthermore, we investigated whether inhibition of miR-188-5p could suppress AAA progression. Experimental AAAs were created in 9-12-week-old male C57BL/6J mice by transient intra-aortic infusion of porcine pancreatic elastase. Expression of miR-188-5p levels were assessed in aneurysmal and control aortae during the progression of aneurysm. For inhibition experiment, miR-188 inhibiting group mice were injected with AAV2-miR188-5p sponge through tail vein and control group mice were injected with AAV2-CMV-GFP. Influences on experimental AAA progression were assessed by measurements of aortic diameter and histopathologic analysis at sacrifice. Meanwhile, immunohistochemistry and fluorescence in situ hybridization were used to determine the inflammatory cells infiltration and colocalization of miR-188-5p in aortic sections. Expression of miR-188-5p is upregulated during progression of AAA. Importantly, miR-188-5p inhibition treatment prevented enlargement of experimental aneurysms. Meanwhile, miR-188-5p inhibition regimens attenuated medial elastin degradation, smooth muscle cell depletion, and mural angiogenesis and the accumulation of macrophages, T cells, and angiogenesis. Furthermore, colocalization of miR188-5p with CD68 and CD3 was observed, which suggest miR-188-5p was expressed mainly in infiltrated macrophages and T cells. Expression of miR-188-5p is increased in experimental AAAs. Treatment with miR-188-5p inhibition limits experimental AAA progression, with histologic evidence of reduced neovessels and attenuated mural leukocyte infiltration. These findings underscore the potential significance of miR-188-5p in aneurysm pathogenesis and as a target for suppression of AAA disease.

Daphnetin suppresses experimental abdominal aortic aneurysms in mice via inhibition of aortic mural inflammation.

Rupture of abdominal aortic aneurysm (AAA) is a devastating event that can be prevented by inhibiting the growth of small aneurysms. Therapeutic strategies targeting certain events that promote the development of AAA must be developed, in order to alter the course of AAA. Chronic inflammation of the aortic mural is a major characteristic of AAA and is related to AAA formation, development and rupture. Daphnetin (DAP) is a coumarin derivative with anti-inflammatory properties that is extracted from Daphne odora var. However, the effect of DAP on AAA development remains unclear. The present study investigated the effect of DAP on the formation and development of experimental AAAs and its potential underlying mechanisms. A mice AAA model was established by intra-aortic infusion of porcine pancreatic elastase (PPE), and mice were intraperitoneally injected with DAP immediately after PPE infusion. The maximum diameter of the abdominal aorta was measured by ultrasound system, and aortic mural changes were investigated by Elastica van Gieson (EVG) staining and immunohistochemical staining. The results demonstrated that DAP significantly suppressed PPE-induced AAA formation and attenuated the depletion of aortic medial elastin and smooth muscle cells in the media of the aorta. Furthermore, the density of mural macrophages, T cells and B cells were significantly attenuated in DAP-treated AAA mice. In addition, treatment with DAP resulted in a significant reduction in mural neovessels. These findings indicated that DAP may limit the formation and progression of experimental aneurysms by inhibiting mural inflammation and angiogenesis. These data confirmed the translational potential of DAP inclinical AAA inhibition strategies.

Spermidine Suppresses Development of Experimental Abdominal Aortic Aneurysms.

BackgroundThe protective effects of polyamines on cardiovascular disease have been demonstrated in many studies. However, the roles of spermidine, a natural polyamine, in abdominal aortic aneurysm (AAA) disease have not been studied. In this study, we investigated the influence and potential mechanisms of spermidine treatment on experimental AAA disease.Methods and ResultsExperimental AAAs were induced in 8‐ to 10‐week‐old male C57BL/6J mice by transient intra‐aortic infusion of porcine pancreatic elastase. Spermidine was administered via drinking water at a concentration of 3 mmol/L. Spermidine treatment prevented experimental AAA formation with preservation of medial elastin and smooth muscle cells. In immunostaining, macrophages, T cells, neutrophils, and neovessels were significantly reduced in aorta of spermidine‐treated, as compared with vehicle‐treated elastase‐infused mice. Additionally, flow cytometric analysis showed that spermidine treatment reduced aortic leukocyte infiltration and circulating inflammatory cells. Furthermore, we demonstrated that spermidine treatment promoted autophagy‐related proteins in experimental AAAs using Western blot analysis, immunostaining, and transmission electron microscopic examination. Autophagic function was evaluated for human abdominal aneurysmal and nonaneurysmal adjacent aortae from AAA patients using Western blot analysis and immunohistochemistry. Dysregulated autophagic function, as evidenced by increased SQSTM1/p62 protein and phosphorylated mTOR, was found in aneurysmal, as compared with nonaneurysmal, aortic segments.ConclusionsOur results suggest that spermidine supplementation limits experimental AAA formation associated with preserved aortic structural integrity, attenuated aortic inflammatory infiltration, reduced circulating inflammatory monocytes, and increased autophagy‐related proteins. These findings suggest that spermidine may be a promising treatment for AAA disease.

Ulinastatin Inhibits the Formation and Progression of Experimental Abdominal Aortic Aneurysms

Aims: Aortic mural inflammatory damage takes a vital part in abdominal aortic aneurysm (AAA). Recently, ulinastatin (UTI) has attracted attention for its anti-inflammatory function. Our study aimed to evaluate potential influences of UTI on experimental AAA. Methods: A mouse model of AAA was induced by infusion of porcine pancreatic elastase (PPE) into the abdominal aorta. Mice were treated with UTI (50,000 U/kg/day i.p.) beginning either immediately or on the 4th day after PPE infusion, with treatment being continued until the 14th day. UTI effects were assessed by aortic diameter measurements and aortic histopathological analysis. Results: Significant and time-dependent aortic diameter enlargement persisted in the control mice from day 0. In the UTI group, aortic diameter increased, and depletion of aortic mural smooth muscle cells and elastin was significantly ­attenuated. Simultaneously, mural CD68+ macrophages, CD8+ T-cell and B220+ B-cell density, as well as neoangiogenesis were suppressed by UTI. In addition, delayed UTI treatment could still effectively limit aneurysm expansion. Conclusions: UTI treatment limits the formation and growth of experimental AAA, and UTI may be a potential treatment for early AAA disease.

Effects of Iliac Stenosis on Abdominal Aortic Aneurysm Formation in Mice and Humans

Reduced lower-limb blood flow has been shown to lead to asymmetrical abdominal aortic aneurysms (AAAs) but the mechanism of action is not fully understood. Therefore, small animal ultrasound (Vevo2100, FUJIFILM VisualSonics) was used to longitudinally study mice that underwent standard porcine pancreatic elastase (PPE) infusion (n = 5), and PPE infusion with modified 20% iliac artery stenosis in the left (n = 4) and right (n = 5) iliac arteries. Human AAA computed tomography images were obtained from patients with normal (n = 9) or stenosed left (n = 2), right (n = 1), and bilateral (n = 1) iliac arteries. We observed rapid early growth and rightward expansion (8/9 mice) in the modified PPE groups (p < 0.05), leading to slightly larger and asymmetric AAAs compared to the standard PPE group. Further examination showed a significant increase in TGFβ1 (p < 0.05) and cellular infiltration (p < 0.05) in the modified PPE group versus standard PPE mice. Congruent, yet variable, observations were made in human AAA patients with reduced iliac outflow compared to those with normal iliac outflow. Our results suggest that arterial stenosis at the time of aneurysm induction leads to faster AAA growth with aneurysm asymmetry and increased vascular inflammation after 8 weeks, indicating that moderate iliac stenosis may have upstream effects on AAA progression.

Abstract 713: Angiotensin 1-7 Suppresses Experimental Abdominal Aortic Aneurysms

Objectives: Abdominal aortic aneurysm (AAA) is a chronic inflammatory disease, the pathogenesis of which remains incompletely defined. We and others have demonstrated the critical role of angiotensin (Ang) II and its receptor AT1 in experimental AAAs. Although Ang 1-7 and its receptor Mas counteract many AT1 functions, the role of the Ang 1-7/Mas pathway has not been studied in AAAs. This study evaluated the influences of Ang 1-7 and Mas on experimental AAAs. Methods: AAAs were created in male C57BL/6J mice via porcine pancreatic elastase (PPE) infusion. Mice were treated with Ang 1-7 (0.5 mg/kg/day via s.c) or vehicle starting immediately prior to, or 4 days following, PPE infusion. Additional mice were co-treated with Ang 1-7 and the Mas receptor antagonist A779 (0.5 mg/kg/day) beginning immediately prior to PPE infusion. Influences on AAAs were evaluated by serial ultrasound imaging and histology. Results: Figure summarizes major results. In ultrasound imaging, at 14 days Ang 1-7 treatment initiated immediately following aneurysm creation substantially suppressed aortic enlargement as compared to vehicle treatment. Histologically, Ang 1-7 treatment was associated with attenuation of aortic medial elastin and smooth muscle cell depletion, mural macrophage, CD4 + , CD8 + T and B cell density and neoangiogenesis. Co-treatment with Ang 1-7 receptor Mas antagonist A779 “rescued” the aneurysm phenotype. Additionally, Ang 1-7 treatment initiated even 4 days following AAA creation was still effective in limiting further aneurysmal enlargement and pathologic evolution. Conclusions: Ang 1-7 treatment, initiated either at, or up to 4 days following, AAA initiation limits further progression of experimental AAAs. This inhibitory effect seems likely related to activation of the Mas receptor. These results suggest that either Ang 1-7 or alternative Mas receptor agonists may have clinical utility in suppressing progression of early AAA disease.

Abstract 058: Efficacy and Mechanisms of Metformin Therapy in Established Experimental Abdominal Aortic Aneurysms

Objective: Diabetes reduces the risk for abdominal aortic aneurysm (AAA) disease. In prior work in rodent models, pre-treatment with metformin was protective against experimental AAA initiation and progression. This study examined the influence of metformin therapy on progression of existing experimental AAAs. Methods: AAAs were created in 10-12 week old male C57BL/6J mice via transient intra-aortic infusion of porcine pancreatic elastase (PPE). Mice were treated with metformin (250 mg/kg via oral gavage), metformin plus AMPK inhibitor Compound C (the later via 10 mg/kg intraperitoneal injection) or vehicle alone for 10 days starting on day 4 following AAA creation. Outcomes were assessed by serial transabdominal ultrasonographic assessment of aortic diameter during treatment, as well as histological examination at sacrifice. Results: The principal findings are summarized in the Figure. Vehicle-treated mice experienced progressive, time-dependent aortic enlargement from day 3 to 14 following PPE infusion. Metformin treatment substantially attenuated further enlargement of existing AAAs. Treatment with Compound C partially rescued the AAA phenotype in metformin-treated mice. Histologically, characteristic aneurysmal pathologic changes, including medial elastin degradation, smooth muscle cell (SMC) depletion, mural leukocyte accumulation and neoangiogenesis (assessed by CD31 staining), present in PPE-infused, vehicle-treated mice, were substantially attenuated with metformin treatment. Co-treatment with Compound C abrogated the effects of metformin on media elastin degradation, SMC depletion and mural macrophage infiltration, with less influence on mural angiogenesis. Conclusion: Metformin therapy suppresses further expansion of existing experimental AAAs. This effect is abrogated by co-treatment with Compound C. These findings further justify performance of a clinical trial of metformin for medical management of AAA disease.

Hypoxia-inducible factor 1 in clinical and experimental aortic aneurysm disease

ObjectiveMural angiogenesis and macrophage accumulation are two pathologic hallmarks of abdominal aortic aneurysm (AAA) disease. The heterodimeric transcription factor hypoxia-inducible factor 1 (HIF-1) is an essential regulator of angiogenesis and macrophage function. In this study, we investigated HIF-1 expression and activity in clinical and experimental AAA disease. MethodsHuman aortic samples were obtained from 24 AAA patients and six organ donors during open abdominal surgery. Experimental AAAs were created in 10-week-old male C57BL/6J mice by transient intra-aortic infusion of porcine pancreatic elastase (PPE). Expression of HIF-1α and its target gene messenger RNA (mRNA) levels were assessed in aneurysmal and control aortae. The HIF-1α inhibitors 2-methoxyestradiol and digoxin, the prolyl hydroxylase domain-containing protein (PHD) inhibitors cobalt chloride and JNJ-42041935, and the vehicle alone as control were administered daily to mice at varying time points beginning before or after PPE infusion. Influences on experimental AAA formation and progression were assessed by serial transabdominal ultrasound measurements of aortic diameter and histopathologic analysis at sacrifice. ResultsThe mRNA levels for HIF-1α, vascular endothelial growth factor A, glucose transporter 1, and matrix metalloproteinase 2 were significantly increased in both human and experimental aneurysm tissue. Tissue immunostaining detected more HIF-1α protein in both human and experimental aneurysmal aortae compared with respective control aortae. Treatment with either HIF-1α inhibitor, beginning before or after PPE infusion, prevented enlargement of experimental aneurysms. Both HIF-1α inhibition regimens attenuated medial elastin degradation, smooth muscle cell depletion, and mural angiogenesis and the accumulation of macrophages, T cells, and B cells. Whereas mRNA levels for PHD1 and PHD2 were elevated in experimental aneurysmal aortae, pharmacologic inhibition of PHDs had limited effect on experimental aneurysm progression. ConclusionsExpression of HIF-1α and its target genes is increased in human and experimental AAAs. Treatment with HIF-1α inhibitors limits experimental AAA progression, with histologic evidence of attenuated mural leukocyte infiltration and angiogenesis. These findings underscore the potential significance of HIF-1α in aneurysm pathogenesis and as a target for pharmacologic suppression of AAA disease.

Inhibition of Phosphatidylinositol 3-kinease suppresses formation and progression of experimental abdominal aortic aneurysms

Accumulating evidence suggests an important role of Phosphatidylinositol 3-kinease (PI3K) pathway in inflammatory cells infiltration. Given the essential role of inflammatory cells infiltration during the formation and progression of abdominal aortic aneurysm (AAA), to investigate the possibility of preventing AAA formation and progression via targeting PI3K is anticipated. Here, experimental AAAs was created in rats by transient intraluminal porcine pancreatic elastase (PPE) infusion into the infrarenal aorta firstly. AAAs rats were administrated with vehicle or Wortmannin during the period of day 0 to day 28 after PPE infusion. The aortic diameter of rats treated with Wortmannin was significantly smaller than those treated with vehicle. Meanwhile, Elastin destruction score and SMC destruction score were significantly decreased in rats treated with Wortmannin. Furthermore, histological analysis revealed infiltration of inflammatory cells were significantly reduced in rats treated with Wortmannin. Finally, the mRNA expression of PI3K and protein expression of pAKT in human abdominal aneurismal aorta tissues was elevated as compare to normal aorta. Our study revealed that PI3K inhibitor suppresses experimental AAAs formation and progression, through mechanisms likely related to impairing inflammation cells infiltration and median elastin degradation. These findings indicated that PI3K inhibitor may hold substantial translation value for AAA diseases.