Rat Abdominal Aortic Aneurysm Model Research Articles

A Novel Method for the Rat Model of Abdominal Aortic Aneurysm Induced by Retroperitoneal Implantation of an Osmotic Pump System With Lipopolysaccharide

Few methods can cocurrently mimic the pathological characteristics and nature history of human abdominal aortic aneurysms (AAAs), especially for the exist of the self-healing tendency of rodents. This study tested a novel method for the AAA rat model induced by retroperitoneal implantation of an osmotic pump system with lipopolysaccharide (LPS) based on the hypothesis that chronic inflammation of perivascular adipose tissue directly influenced the development and progression of AAAs. 20 male Sprague-Dawley rats (10-month-old) fed with the Paigen diet were randomly divided into 4 groups: the blank group ×2, the sham group ×4, the empty capsule group ×4, and the LPS capsule group ×10. The LPS capsule group received implantations of the ALZET® osmotic pump capsule with LPS (3.6μg/day) parallel to the abdominal aorta through a retroperitoneal approach. Two weeks later, 6 rats were randomly selected from the LPS capsule group to form the anti-inflammatory group and received implantations of another osmotic pump capsule with interleukin (IL)-10 (75ng/day) through the same approach. The changes in abdominal aortic diameter were observed by ultrasound every 2weeks, and samples were harvested for histopathologic and immunohistochemical analysis 6weeks later. Within the 6weeks after modeling, the LPS capsule group showed sustained and significant aortic dilatation (P<0.01), while the anti-inflammatory group showed a rapid and obvious shrinkage 2weeks after the IL-10 osmotic pump capsule implantation (P<0.01). The LPS capsule group presented excellent pathological mimicking of human AAAs and showed severe medial degeneration with the least elastic content among the 5 groups at the end of the sixth week (P<0.05). Notably, the anti-inflammatory group showed perfect medial preservation with the most elastic content (P<0.05) and the highest elastin/collagen ratio (P<0.01) at the end of the study. Matrix metalloproteinases (MMP) 2 and 9 and toll-like receptor 2 showed strong expression in the LPS capsule group at the end of the sixth week, which was significantly higher than that in the blank group and sham group. Interestingly, the anti-inflammatory group showed a slightly higher MMP9 expression than the LPS capsule group though there was no statistical difference between them. This novel method for the rat AAA model induced by retroperitoneal implantation of an osmotic pump capsule with LPS can concurrently mimic the histological characteristics and natural history of human AAAs. Further studies were needed to improve the osmotic pump system.

Dexmedetomidine Alleviates Abdominal Aortic Aneurysm by Activating Autophagy Via AMPK/mTOR Pathway.

Abdominal aortic aneurysms (AAA) are a critical global health issue with increasing prevalence. Dexmedetomidine (DEX) is a highly selective α2-adrenoceptor agonist that has previously been shown to play a protective role in AAA. Nevertheless, the mechanisms underlying its protection effect remain not fully understood. A rat AAA model was established via intra-aortic porcine pancreatic elastase perfusion with or without DEX administration. The abdominal aortic diameters of rats were measured. Hematoxylin-eosin and Elastica van Gieson staining were conducted for histopathological observation. TUNEL and immunofluorescence staining were utilized to detect cell apoptosis and α-SMA/LC3 expression in the abdominal aortas. Protein levels were determined using western blotting. DEX administration repressed the dilation of aortas, alleviated pathological damage and cell apoptosis, and suppressed phenotype switching of vascular smooth muscle cells (VSMCs). Moreover, DEX activated autophagy and regulated the AMP-activated protein kinase/mammalian target of the rapamycin (AMPK/mTOR) signaling pathway in AAA rats. Administration of the AMPK inhibitor attenuated the DEX-mediated ameliorative effects on AAA in rats. DEX ameliorates AAA in rat models by activating autophagy via the AMPK/mTOR pathway.

Establishment of a New Abdominal Aortic Aneurysm Model in Rats by a Retroperitoneal Approach.

BackgroundConstructing an ideal model of abdominal aortic aneurysm (AAA) is of great significance to elucidate its complex pathogenesis. Therefore, we introduce a new and simple method to simulate human AAA and construct a rat AAA model through a retroperitoneal approach.MethodsForty healthy adult Sprague Dawley (SD) rats were randomly divided into a control group, elastase + calcium chloride group (PPE+CaCl2), elastase group (PPE), and elastase + beta aminopropionitrile group (PPE+BAPN) according to a male-female ratio of 1:1, with 10 rats in each group. A retroperitoneal approach was used to free the infrarenal abdominal aorta in all four groups. In the PPE + CaCl2 group, 0.1 ml of elastase (approximately 5 U) was perfused into the arterial cavity for 20 min, and 1.0 mol/L calcium chloride was infiltrated out of the arterial cavity for 10 min. In the PPE group, 0.1 mL of elastase (approximately 5U) was perfused into the arterial cavity for 20 min, and normal saline was infiltrated out of arterial cavity for 10 min; the PPE + BAPN group combined with 0.3% BAPN drinking water/day on the basis of PPE group; the control group was treated with saline instead of elastase and calcium chloride. Abdominal aortic specimens were collected after 4 weeks of feeding. The diagnostic criteria of AAA were 50% dilation of the abdominal aorta or rupture of the aneurysm at 4 weeks after the operation. Histopathology, immunohistochemistry, quantitative PCR (qPCR), western blotting assay, gelatine zymogram, and other methods were used.ResultsThe operation time of the four groups was controlled at approximately 40 min, and the success rate of the operation was 100%. Survival rate: Control Group (100%) = PPE Group (100%) > PPE + CaCl2 Group (90%) > PPE + BAPN Group (40%); Aneurysm formation rate: PPE + BAPN Group (100%) > PPE + CaCl2 Group (80%) > PPE Group (60%) > Control Group (0%); Aneurysm rupture rate: PPE + BAPN group (60%) > PPE + CaCl2 group (12.5%) > PPE group (0%);Inflammatory cells (macrophages, T cells, B cells, dendritic cells) infiltrated in different degrees in the PPE + CaCl2, PPE and PPE + BAPN groups. Vascular thickness, elastic fiber content, collagen fiber content, and vascular smooth muscle cell content in the PPE + CaCl2 group and PPE + BNPA group were significantly lower than those in Control group (P < 0.05). The content of elastic fibers and vascular smooth muscle cells in the PPE group were significantly lower than that in Control group (P < 0.05). The expression and activity of matrix metalloproteinase 2 (MMP2) and MMP9 in the PPE + CaCl2 group, PPE group, and PPE + BNPA group were significantly higher than those in the control group (P < 0.05).ConclusionsA new, simple, and reproducible rat AAA model can be constructed by a retroperitoneal approach. The pathological features of the three models are effective simulation of human AAA inflammatory cell infiltration, protease activity enhancement, and extracellular matrix destruction. The PPE+ CaCl2 model has the advantages of a high survival rate, high aneurysm formation rate, good stability, and reproducibility. It is an ideal animal model for studying the pathogenesis of AAA. The PPE + BAPN model can simulate the characteristics of spontaneous rupture of aneurysms. It is an ideal animal model to study the mechanism of AAA rupture.

Assessing the targeting and fate of cathepsin k antibody-modified nanoparticles in a rat abdominal aortic aneurysm model

Abdominal aortic aneurysms (AAAs), a prototypic proteolytic cardiovascular disorder, are localized expansions of the aortal wall. Chronically upregulated and overexpressed proteases irreversibly degrade and disrupt the elastic matrix, which provides stretch and recoil properties to the aortal wall. Adult vascular smooth muscle cells are inherently unable to produce sufficient elastin to form new elastic fibers to naturally repair the aortal wall and the AAA continues to grow until fatal rupture. Surgical intervention is reserved for AAAs with a high risk of rupture, but there is currently no treatment for small, still growing AAAs. We have previously developed matrix regenerative PEG-PLGA nanoparticles (NPs) with pro-elastogenic and anti-proteolytic properties that act synergistically with a released therapeutic. However, strategies are required to effectively deliver these NPs to the disease site to avail of these benefits. We have identified cathepsin K, a protease overexpressed in AAA tissue, as a potential substrate for antibody based active targeting. We sought to assess the safety and biocompatibility of NPs with anti-cathepsin K antibodies conjugated to the NP surface (cat K Ab-NPs) and then assess their biodistribution and retention in both the targeted aorta and non-target organs in a rat AAA model. In this work, we show that cat K Ab-NPs can selectively target the aneurysmal aorta in a rat AAA model. However, there is unwanted NP uptake and retention in non-target organs that can be addressed in future work. Still, cathepsin K is a viable target for active delivery of NPs in an AAA model. Statement of SignificanceWe have previously developed elastic matrix regenerative polymer nanoparticles (NPs), but require strategies to efficiently target the disease site. Antibodies against cathepsin K, an overexpressed protease in abdominal aortic aneurysms, have been conjugated to the NP surface to act as a targeting moiety. In this work, we assessed NP safety and in vivo biodistribution in an aneurysmal rat model and demonstrated positive targeting and retention for up to 2 weeks within the aortal wall.

Myeloid related protein 8/14 is a new candidate biomarker and therapeutic target for abdominal aortic aneurysm

Open surgical or percutaneous endovascular (EVAR) intervention is the only approved treatment for abdominal aortic aneurysm (AAA). Novel targeted therapy and companion diagnostic are needed. Our study aimed to investigate the expression of myeloid related protein 8/14 (MRP8/14) in AAA and explore whether MRP8/14 can be a biomarker and therapeutic target for AAA. The serum levels of MRP8/14 and inflammatory factors including TNF-α, IL-1β, and IL-6 were increased in 20 human AAA patients compared to healthy people by ELISA assay. The mRNA and protein expressions of MRPs in AAA tissues of AAA patient were significantly elevated when compared with the levels in adjacent non-aneurysmal aortic segments in AAA patients. through PCR and western blot analysis. Correlation analysis and receiver operating characteristic (ROC) curve analysis confirmed that MRP8/14 was secreted into the serum and possessed the potential diagnostic value. Rat AAA models established by perfusion porcine pancreatic elastase and murine macrophage RAW264.7 cells were used to evaluate the mechanisms of MRP8/14 after treatment with antibodies. Similarly, MRP8, MRP14, and MRP8/14 were highly expressed in rat AAA model, while the administrations of antibodies of MRPs significantly reversed the improvement expressions of MRP8 and MRP14. In RAW264.7 cells, MRPs especially for MRP8/14 obviously increased the levels of MMP-2 and MMP-9. Under MRP8/14 stimulation, the antibodies of MRPs recovered the expressions of MMP-2 and MMP-9. Anti MRP8/14 antibody exhibited the strongest effect in the experiments. Our results indicated that MRP8/14 was associated with AAA presence and progression and could be considered as a biomarker for AAA diagnose. Anti MRP8/14 can be a potential therapeutic target for treatment of AAA.

Pentagalloyl Glucose and Its Functional Role in Vascular Health: Biomechanics and Drug-Delivery Characteristics.

Pentagalloyl glucose (PGG) is an elastin-stabilizing polyphenolic compound that has significant biomedical benefits, such as being a free radical sink, an anti-inflammatory agent, anti-diabetic agent, enzymatic resistant properties, etc. This review article focuses on the important benefits of PGG on vascular health, including its role in tissue mechanics, the different modes of pharmacological administration (e.g., oral, intravenous and endovascular route, intraperitoneal route, subcutaneous route, and nanoparticle based delivery and microbubble-based delivery), and its potential therapeutic role in vascular diseases such as abdominal aortic aneurysms (AAA). In particular, the use of PGG for AAA suppression and prevention has been demonstrated to be effective only in the calcium chloride rat AAA model. Therefore, in this critical review we address the challenges that lie ahead for the clinical translation of PGG as an AAA growth suppressor.

The Effect of Pentagalloyl Glucose on the Wall Mechanics and Inflammatory Activity of Rat Abdominal Aortic Aneurysms.

An abdominal aortic aneurysm (AAA) is a permanent localized expansion of the abdominal aorta with mortality rate of up to 90% after rupture. AAA growth is a process of vascular degeneration accompanied by a reduction in wall strength and an increase in inflammatory activity. It is unclear whether this process can be intervened to attenuate AAA growth, and hence, it is of great clinical interest to develop a technique that can stabilize the AAA. The objective of this work is to develop a protocol for future studies to evaluate the effects of drug-based therapies on the mechanics and inflammation in rodent models of AAA. The scope of the study is limited to the use of pentagalloyl glucose (PGG) for aneurysm treatment in the calcium chloride rat AAA model. Peak wall stress (PWS) and matrix metalloproteinase (MMP) activity, which are the biomechanical and biological markers of AAA growth and rupture, were evaluated over 4 weeks in untreated and treated (with PGG) groups. The AAA specimens were mechanically characterized by planar biaxial tensile testing and the data fitted to a five-parameter nonlinear, hyperelastic, anisotropic Holzapfel–Gasser–Ogden (HGO) material model, which was used to perform finite element analysis (FEA) to evaluate PWS. Our results demonstrated that there was a reduction in PWS between pre- and post-AAA induction FEA models in the treatment group compared to the untreated group using either animal-specific or average material properties. However, this reduction was not statistically significant. Conversely, there was a statistically significant reduction in MMP-activated fluorescent signal between pre- and post-AAA induction models in the treated group compared to the untreated group. Therefore, the primary contribution of this work is the quantification of the stabilizing effects of PGG using biomechanical and biological markers of AAA, thus indicating that PGG could be part of a new clinical treatment strategy that will require further investigation.

Suppression of aortic expansion and contractile recovery in a rat abdominal aortic aneurysm model by biodegradable gelatin hydrogel sheet incorporating basic fibroblast growth factor.

Biodegradable gelatin hydrogel sheet (BGHS) incorporating basic fibroblast growth factor (bFGF) may inhibit the progression of abdominal aortic aneurysm (AAA). We investigated whether AAA in a rat model treated with BGHS soaked with bFGF can suppress aortic expansion and recover the contractile response of aneurysmal aortic wall. Experimental AAA was induced in 10-week-old male Sprague-Dawley rats with intra-aortic elastase infusion. Aortas of these rats were assigned to 4 groups (n=6 each) as follows: Control group, aortas infused with saline; Elastase only group, aortas infused with elastase; Hydrogel group, aortas wrapped with saline-soaked BGHS after elastase infusion; and bFGF group, aortas wrapped with bFGF (100μg)-soaked BGHS after elastase infusion. Preoperatively and on postoperative day (POD)7 and POD14, mean aortic maximal diameter was measured ultrasonographically. Aortic expansion ratio was calculated as: (post-infusion aortic diameter on POD14/pre-infusion aortic diameter×100). Aortas were stained with Elastica van Gieson and α-smooth muscle actin to measure the ratio of elastic fibers and α-smooth muscle actin-positive cells area to the media area. Aortas on POD14 were cut into 2-mm rings and treated with contractile agent, then tension was recorded using myography. Maximum aorta diameters were significantly greater in Elastase only group, Hydrogel group, and bFGF group than in Control group (on POD14). Maximum diameter was significantly lower in bFGF group (3.52±0.4mm) than in Elastase only group (6.21±1.4mm on POD14, P<.05). On histological analysis, ratio of the area staining positively for elastic fibers was significantly greater in bFGF group (7.43±1.8%) than in Elastase only group (3.76±2.9%, P<.05). The ratio for α-smooth muscle actin-positive cells was significantly lower in Elastase only group (38.3±5.1%) than in Control group (49.8±6.7%, P<.05). No significant differences were seen between Elastase only group and bFGF group, but ratios tended to be increased in bFGF group. Consecutive mean contractile tensions were significantly higher in bFGF group than in Elastase only group. Maximum contractile tension was significantly higher in bFGF group (1.3±0.4mN) than in Elastase only group (0.4±0.2mN, P<.05). Aortic expansion can be suppressed and contractile responses of aneurysmal aortic wall recovered using BGHS incorporating bFGF.

A Novel Modification of the Murine Elastase Infusion Model of Abdominal Aortic Aneurysm Formation

To create a novel procedure that will decrease the mortality of experimental animals in the intraarterial infusion of elastase abdominal aortic aneurysm (AAA) model. Novel models were created by means of direct puncture in the infrarenal abdominal aortic aorta, intraluminal elastase in the 1-cm segment of abdominal aorta. Femoral artery cannula approach and infusing with elastase was considered as the traditional group and that infusing with saline solution as the control group. Survival rate, morphology and histology of aneurysms, and inflammation mediators were calculated. Among the 36 rats, the average length from testicular arteries to left iliolumbar artery was 1.18±0.22cm, and 77.8% of them were longer than 1cm. Procedure time was significantly shorter in novel group than that in 2 other groups (P=0.006; P<0.0001). During 24hr postoperation, no death was observed in the novel group. Within 4 wk, survival rate in the control group was 60.6% and 80.8% in the novel group whereas 41.0% in the traditional group. Till the second week, all rats in the traditional and novel group had formed AAAs. And then, the survival rates and rupture rates of AAA between the 2 groups were similar within the following 2 wk (P=0.487; P=0.539). Inflammation degree and elastase content in intima media of aneurysms were similar (P=0.720). However, Tumor necrosis factor alpha and Interleukin-1 beta levels were significantly lower in the novel group than those in the traditional group (P<0.0001; P<0.0001). A novel rat AAA model was created by intraluminal elastase infusion through direct puncture the infrarenal aorta. This model is efficient and reliable, with a high survival rate and with similar morphology and histology of aortic aneurysms.

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.

Expression of chemokine receptor-4 in bone marrow mesenchymal stem cells on experimental rat abdominal aortic aneurysms and the migration of bone marrow mesenchymal stem cells with stromal-derived factor-1

This study investigated the expression and role of chemokine receptor-4 (CXCR4) in bone marrow mesenchymal stem cells (BMSCs) from experimental rats with abdominal aortic aneurysms (AAA) for migration of BMSCs. Sprague–Dawley rats were divided into an experimental group and control group (n = 18 each). AAA was induced with 0.75 M solution infiltrate for 30 minutes, after which the abdomen was rinsed and closed. Saline was used in place of CaCl2 in the control group. CD34 and CD29 were detected by flow cytometry, the gene and protein expression of CXCR4 were detected by real-time polymerase chain reaction and western blot, respectively. The migration of BMSCs with stromal-derived factor-1 was detected by Transwell chamber. CD34 expression was negative and CD29 expression was positive. The gene and protein expression of CXCR4 were significantly higher in experimental group than them in control group (p < 0.05), the migration ability of BMSCs from the experimental group was significantly higher than that from the control group (p < 0.05). Stromal-derived factor -1/CXCR4 can enhance the migration of BMSCs in vitro in a rat AAA model.

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.

Orally administered dipeptidyl peptidase-4 inhibitor (alogliptin) prevents abdominal aortic aneurysm formation through an antioxidant effect in rats

Dipeptidyl peptidase-4 (DPP-4) inhibitor, a novel antidiabetic drug, has a cardioprotective effect on ischemia-reperfusion injury through an antioxidant effect. However, the effect of DPP-4 inhibitor on aneurysm formation has not been investigated. We aimed to test the hypothesis that the DPP-4 inhibitor, alogliptin, attenuates vascular oxidative stress and thus inhibits abdominal aortic aneurysm (AAA) formation. AAAs were created with intraluminal elastase and extraluminal calcium chloride in 36 male rats. Rats were divided into three groups: a low dose of alogliptin group (group LD; 1 mg/kg/d), a high-dose group (group HD; 3 mg/kg/d), and a control group (group C, water). Alogliptin was administered by gastric gavage once daily beginning 3 days before surgery. On day 7 after aneurysm preparation, reactive oxygen species (ROS) expression was semiquantified by dihydroethidium staining, and the oxidation product of DNA produced by ROS, 8-hydroxydeoxyguanosine (8-OHdG), was measured by immunohistochemical staining. Blood glucose concentrations were measured. Hematoxylin and eosin and elastica Van Gieson stainings were performed on day 28, and the AAA dilatation ratio was calculated. On day 7 (six in each group), dihydroethidium staining of the aneurysm wall showed a reduced level of ROS expression (4.6 ± 0.6 in group C, 2.7 ± 0.3 in group LD, and 1.7 ± 0.5 in group HD; P < .0001) and showed fewer 8-OHdG-positive cells in alogliptin-treated samples (138.1 ± 7.4 cells in group C, 102.5 ± 4.5 cells in group LD, and 66.1 ± 4.5 cells in group HD; P < .0001) The treatment significantly reduced messenger RNA expression of matrix metalloproteinases (MMPs) in aneurysm walls (relative expression: MMP-2: 2.1 ± 0.4 in group C, 1.3 ± 0.3 in group LD, and 0.9 ± 0.2 in group HD; P < .001; MMP-9: 2.0 ± 0.5 in group C, 0.3 ± 0.3 in group LD, and 0.3 ± 0.2 in group HD; P < .001). On day 28 (six in each group), the aortic wall in groups LD and HD was less dilated (dilatation ratio: 199.2% ± 11.8% in group C, 159.6% ± 2.8% in group LD, and 147.1% ± 1.9% in group HD; P < .02 group C vs HD) and had higher elastin content than in group C. The difference in blood glucose levels among the three groups was not significant. The DPP-4 inhibitor, alogliptin, attenuates aneurysm formation and expansion dose-dependently in a rat AAA model via an antioxidative action.

Correlation of osteopontin and nuclear factor-κB expression with pathogenesis of abdominal aortic aneurysm in rats

Objective To establish rat model of abdominal aortic aneurysm (AAA),observe the expression of osteopontin (OPN) and nuclear factor-κB (NF-κB) and explore the molecular mechanism of AAA.Methods Thirty rats were divided into 3 groups (n =10 each).The rat AAA models were made by perfusing pancreatopeptidase E from hog pancreas into the aorta of rats for 30 min.Aortas were taken out two weeks after perfusion and the diameter of all aortas was measured.Hematoxylin and eosin (HE) staining was used to observe the structural change and elastase staining to observe how the area of elastase altered in the middle tuna of the aorta.Immunohistochemistry and Western blotting were used to detect the expression of OPN,NF-κB and matrix metalloproteinase-2 (MMP-2).Results Rat AAA model was successfully made.As compared with saline group and normal group,the infiltration of inflammatory cells was significantly increased and the quantity of elastase was significantly reduced in experimental group.Immunohistochemistry revealed that the expression of OPN was significantly increased in experimental group (P ＜ 0.05).Western blotting demonstrated that the expression of OPN was also obviously increased in experimental group (P ＜ 0.01).Conclusion OPN may increase the expression of MMP-2 through the NF-κB pathway in rat AAA model,thereby accelerating the degradation of extracellular matrix and eventually leading to occurrence of AAA. Key words: Abdominal aortic aneurysm; Osteopontin; Nuclear factor-κB; Matrix metallo-proteinase-2

Free-radical scavenger edaravone inhibits both formation and development of abdominal aortic aneurysm in rats

An ideal pharmaceutical treatment for abdominal aortic aneurysm (AAA) is to prevent aneurysm formation and development (further dilatation of pre-existing aneurysm). Recent studies have reported that oxidative stress with reactive oxygen species (ROS) is crucial in aneurysm formation. We hypothesized that edaravone, a free-radical scavenger, would attenuate vascular oxidative stress and inhibit AAA formation and development. An AAA model induced with intraluminal elastase and extraluminal calcium chloride was created in 42 rats. Thirty-six rats were divided three groups: a low-dose (group LD; 1 mg/kg/d), high-dose (group HD; 5 mg/kg/d), and control (group C, saline). Edaravone or saline was intraperitoneally injected twice daily, starting 30 minutes before aneurysm preparation. The remaining six rats (group DA) received a delayed edaravone injection (5 mg/kg/d) intraperitoneally, starting 7 days after aneurysm preparation to 28 days. AAA dilatation ratio was calculated. Pathologic examination was performed. ROS expression was semi-quantified by dihydroethidium staining and the oxidative product of DNA induced by ROS, 8-hydroxydeoxyguanosine (8-OHdG), by immunohistochemical staining. At day 7, ROS expression and 8-OHdG-positive cells in aneurysm walls were decreased by edaravone treatment (ROS expression: 3.0 ± 0.5 in group LD, 1.7 ± 0.3 in group HD, and 4.8 ± 0.7 in group C; 8-OHdG-positive cells: 106.2 ± 7.8 cells in group LD, 64.5 ± 7.7 cells in group HD, and 136.6 ± 7.4 cells in group C; P < .0001), compared with group C. Edaravone treatment significantly reduced messenger RNA expressions of cytokines and matrix metalloproteinases (MMPs) in aneurysm walls (MMP-2: 1.1 ± 0.5 in group LD, 0.6 ± 0.1 in group HD, and 2.3 ± 0.4 in group C; P < .001; MMP-9: 1.2 ± 0.1 in group LD, 0.2 ± 0.6 in group HD, and 2.4 ± 0.2 in group C; P < .001). At day 28, aortic walls in groups LD and HD were less dilated, with increased wall thickness and elastin content than those in group C (dilatation ratio: 204.7% ± 16.0% in group C, 156.5% ± 6.6% in group LD, 136.7% ± 2.0% in group HD; P < .0001). Delayed edaravone administration significantly prevented further aneurysm dilatation, with increased elastin content (155.2% ± 2.9% at day 7, 153.1% ± 11.6% at day 28; not significant). Edaravone inhibition of ROS can prevent aneurysm formation and expansion in the rat AAA model. Free-radical scavenger edaravone might be an effective pharmaceutical agent for AAA in clinical practice.

Systemic Administration of Ribbon-type Decoy Oligodeoxynucleotide Against Nuclear Factor κB and Ets Prevents Abdominal Aortic Aneurysm in Rat Model

Currently, there is no effective clinical treatment to prevent abdominal aortic aneurysm (AAA). To develop a novel therapeutic approach, we modified decoy oligodeoxynucleotide (ODN) against nuclear factor κB (NFκB) and ets, to a ribbon-shaped circular structure without chemical modification, to increase its resistance to endonuclease for systemic administration. Intraperitoneal administration of ribbon-type decoy ODNs (R-ODNs) was performed in an elastase-induced rat AAA model. Fluorescent isothiocyanate (FITC)-labeled R-ODNs could be detected in macrophages migrating into the aneurysm wall, and NFκB and ets activity were simultaneously inhibited by chimeric R-ODN. Treatment with chimeric R-ODN significantly inhibited aortic dilatation, whereas conventional phosphorothioate decoy ODN failed to prevent aneurysm formation. Significant preservation of elastic fibers was observed with chimeric R-ODN, accompanied by a reduction of secretion of several proteases from macrophages. Activation of matrix metalloproteinase (MMP)-9 and MMP-12, but not MMP-2, was suppressed in the aneurysm wall by chimeric R-ODN, whereas recruitment of macrophages was not inhibited. Treatment with chimeric R-ODN also inhibited the secretion of cathepsin B and K from macrophages. Overall, the present study demonstrated that systemic administration of chimeric R-ODNs prevented aneurysm formation in a rat model. Further modification of the decoy strategy would provide a means of less invasive molecular therapy for human AAA.

Activation of transglutaminase type 2 for aortic wall protection in a rat abdominal aortic aneurysm formation

The altered structure and composition of the vascular extracellular matrix (ECM) influences the formation of abdominal aortic aneurysms (AAA). Transglutaminase type 2 (TG2), which is a Ca(2+)-dependent cross-linking enzyme, has been proven the importance for ECM homeostasis, but there is no evidence of TG2 in AAA formation. The hypothesis was investigated that TG2 contributes to protect aortic walls during remodeling of the AAAs. In a rat abdominal aortic aneurysm model using a combination of intraluminal elastase infusion and extraluminal calcium chloride, TG2 expression and activity were evaluated at 1 and 8 weeks after the AAA preparation (n = 6 at each endpoint), compared with those of the non-prepared aorta (n = 6). Additionally, ex vivo experiments of isolated AAA tissue culture with recombinant human TG2, TG2 inhibitor cystamine, or tissue necrosis factor (TNF)-α were performed. TG2 mRNA expression in the AAAs was significantly upregulated at both 1 and 8 weeks (22.4-fold and 5.4-fold increases of the non-prepared aorta, P = .0022 and P = .0048, respectively). TG2 protein expression and activity were also enhanced by fluorescent staining of the AAAs. Similar mRNA upregulation of TNF-α, interleukin-1β, matrix metalloproteinases (MMP)-2, MMP-9, and tissue inhibitors of metalloproteinases (TIMP)-1 and TIMP-2 was observed in the AAAs, and TG2 and TNF-α were colocalized in the aortic walls at 1 week. Ex vivo experiments showed that mRNA expressions of TNF-α, MMP-2, and MMP-9 in the cultured AAA tissue were decreased by exogenous TG2, whereas were increased by cystamine. TNF-α exposure to the AAA tissues was significantly upregulated TG2 mRNA expression (P = .0333). TG2 expression and activity in AAA formation were enhanced, possibly due to compensatory reaction. TG2 has a potential role of ECM protector in aortic walls during remodeling of the AAAs.

A novel rat model of abdominal aortic aneurysm using a combination of intraluminal elastase infusion and extraluminal calcium chloride exposure

An ideal animal model of abdominal aortic aneurysm (AAA) is of great importance for clarifying unknown complex mechanisms of the pathogenesis. We introduce a new, simple technique to create reliable AAAs that simulate human aneurysms. Experimental models of AAAs were created in 71 rats by means of a 20-minute application of intraluminal elastase (30 U) and extraluminal calcium chloride (0.5M) in the 1-cm segment of infrarenal abdominal aorta (group EC, n = 26). A single application of elastase (group E, n = 24) or calcium chloride (group C, n = 21) was used as control. The treated aorta in each group was measured under physiologic conditions and harvested at 1 and 4 weeks. Successful AAA formation was defined as a dilation ratio >50%. Inflammatory response, elastolytic activity, and histology in the treated aorta were evaluated among the three groups. The surgical procedure in each group was similarly completed for approximate 30 minutes and performed without any technical failure or operative death. At 4 weeks, the dilation ratio and wall thickness were 94.8% +/- 9.9% and 125.4 +/- 5.6 microm in group EC, 43.3% +/- 6.3% and 149.6 +/- 6.5 microm in group E, and 10.9% +/- 4.2% and 152.9 +/- 7.2 microm in group C. The success rate of AAA formation in group EC (92.7%) was significantly higher than that in group E (25.0%) and group C (0.0%). Less elastin content in the aortic wall was observed in group EC. At 1 week, tumor necrosis factor-alpha and interleukin-1beta messenger RNA (mRNA) expressions were significantly upregulated, and CD3+ and CD11b+ cells were significantly infiltrated into the treated aorta of group EC, compared with groups E or C. Gelatinolytic activities and mRNA expressions of matrix metalloproteinase (MMP)-2 and MMP-9 were also significantly activated in group EC. The rat AAA model using a combination of intraluminal elastase infusion and extraluminal calcium chloride exposure is simple and easy to perform and is highly reliable and reproducible to create a saccular aneurysm similar to human AAAs. This model could be more useful to clarify AAA pathogenesis, mechanisms, and treatment interventions in experimental researches.

Abstract 430: Inhibition of Aneurysm Development by Intra-peritoneal Application of Ribbon-type Decoy Oligodeoxynucleotide against Nuclear Factor-κB and Ets in a Rat Model

As elective surgical or endovascular repair is not effective to improve survival of small abdominal aortic aneurysm (AAA), the development of a novel therapeutic approach for treating small AAA is awaited. We have previously reported the regressive effect of chimeric decoy oligodeoxynucleotides (ODN) against NFκB and ets in an experimental AAA model. However, local application of conventional phosphorothioated chimeric decoy ODN (PC-ODN) was necessary due to easily degradation. Therefore, we developed a novel chimeric decoy ODN with a ribbon-shaped circular structure (RC-ODN) to stabilize decoy ODN against nucleases. Transfection of RC-ODN inhibited MMP-2 expression in VSMC, but not MMP-9 expression. In contrast, in cultured THP-1 cells, MMP-9 expression was suppressed by RC-ODN, whereas MMP-2 expression was not altered. Interestingly, these inhibitory effects of RC-ODN were more potent than that of PC-ODN (n=8, P&lt;0.05). Next, intra-peritoneal application of RC-ODN was performed in an elastase-induced rat AAA model. One week after injection, FITC-labeled RC-ODN were detected in migrating macrophages into aneurysm wall. In addition, both NFκB and ets activity were suppressed by RC-ODN. Four weeks after treatment, RC-ODN treatment inhibited the aortic dilatation as compared to control or scrambled decoy ODN transfer (n=6, P&lt;0.05), while PC-ODN treatment failed to prevent aneurysm formation. Interestingly, a significant preservation of elastic fibers was observed with RC-ODN treatment, accompanied by a reduction of expression of MMP-9, MMP-12, cathepsin B and cathepsin K (P&lt;0.05), while the recruitment of macrophages was not inhibited. Finally, to consider the clinical application, we investigated the effect of intravenous injection of RC-ODN. Four weeks after infusion of angiotensin II in apolipoprotein E-deficient mice, intravenous injection of RC-ODN (1 mg/Kg) was performed 3 times per week. RC-ODN treatment showed a tendency to decrease the size of AAA, but not significant. In conclusion, the present study provided a novel strategy to treat AAA using intra-peritoneal application of RC-ODN in a rat model. Further development of this strategy would be useful to treat human AAA, as a minimally invasive molecular therapy.

Inhibition of development of experimental aortic abdominal aneurysm in rat model by atorvastatin through inhibition of macrophage migration

Recently, atherosclerosis has been considered to be the result of inflammation. Interestingly, hydroxymethylglutaryl-coenzyme (HMG-Co) A inhibitors (statins), which are clinically used as lipid-lowering agents, have been reported to have various anti-inflammatory effects. As abdominal aortic aneurysm (AAA) is a common degenerative condition associated with atherosclerosis, this study was designed to investigate the inhibitory effect of a statin, atorvastatin, on aneurysm formation apart from its lipid-lowering effect. We employed an elastase-induced rat AAA model, as statins do not lower cholesterol in rats. Mean aneurysm diameter was significantly smaller in the atorvastatin treatment group as compared to control at 4 weeks after surgery ( P < 0.05). Interestingly, atorvastatin inhibited the expression of ICAM and MCP-1, followed by the suppression of macrophage recruitment into the aortic wall at 1 week after operation. A significant reduction in MMP-12, but not MMP-2, -3 and -9, expression was also observed by treatment with atorvastatin at 1 week after surgery. In addition, synthesis of collagen and elastin in the vascular wall were significantly increased by atorvastatin. Here, the present study demonstrated a direct effect of atorvastatin to inhibit the progression of aortic aneurysm, independent of its lipid-lowering effect. This study suggests new therapeutic aspects of statins to inhibit the progression of aneurysms.