Induced Abdominal Aortic Aneurysm Formation Research Articles

Medium-Dose Formoterol Attenuated Abdominal Aortic Aneurysm Induced by EPO via β2AR/cAMP/SIRT1 Pathway.

Abdominal aortic aneurysm (AAA) is a life-threatening vascular disease but effective drugs for treatment of AAA are still lacking. Recently, erythropoietin (EPO) is reported to induce AAA formation in apolipoprotein-E knock out (ApoE-/-) mice but an effective antagonistis unknown. In this study, formoterol, a β2 adrenergic receptor (β2AR) agonist, is found to be a promising agent for inhibiting AAA. To test this hypothesis, ApoE-/- mice are treated with vehicle, EPO, and EPO plus low-, medium-, and high-dose formoterol, respectively. The incidence of AAA is 0, 55%, 35%,10%, and 55% in these 5groups, respectively. Mechanistically, senescence of vascular smooth muscle cell (VSMC) is increased by EPO while decreased by medium-dose formoterol both in vivo and in vitro, manifested by the altered expression of senescence biomarkers including phosphorylation of H2AXserine139, senescence-associated β-galactosidase activity, and P21 protein level. In addition, expression of sirtuin 1 (SIRT1) in aorta is decreased in EPO-induced AAA but remarkably elevatedby medium-dose formoterol. Knockdown of β2AR and blockage of cyclic adenosine monophosphate (cAMP) attenuate the inhibitory role of formoterol in EPO-induced VSMC senescence. In summary, medium-dose formoterol attenuates EPO-induced AAA via β2AR/cAMP/SIRT1 pathways, which provides a promising medication for the treatment of AAA.

CD147 monoclonal antibody attenuates abdominal aortic aneurysm formation in angiotensin II-Infused apoE-/- mice.

Abdominal aortic aneurysm (AAA) is a life threatening vascular disease. Our previous study reported the upregulation of CD147 expression in human aortic aneurysms. In this study, we injected apoE-/- mice intraperitoneally with CD147 monoclonal antibody or IgG control antibody to observe its effect on Angiotensin II (AngII) induced AAA formation. ApoE-/- mice were randomly divided into an AngⅡ+CD147 antibody group (n=20) and an AngⅡ+IgG antibody group (n=20). The Alzet osmotic minipump was implanted subcutaneously into the backs of mice to infuse AngII (1000ng/kg/min) for 28days and subsequently treated with CD147 monoclonal antibody or control IgG mAb (10μg/mouse/day) beginning one day after surgery. Body weight, food intake, drinking volume and blood pressure were measured weekly throughout the study. After 4weeks of injection, routine bloodwork measuring liver function, kidney function and lipid levels were recorded. Hematoxylin and eosin (H&E), Masson's trichrome, and Elastic van Gieson (EVG) staining were used to evaluate the pathological changes in blood vessels. In addition, Immunohistochemical assay was used to detect infiltration of inflammatory cells. Tandem mass tag (TMT)-based proteomic analysis was used to define differentially expressed proteins (DEPs) using a p-value<0.05 and fold change>1.2 or<0.83 as the threshold. Subsequently, we conducted protein-protein interaction (PPI) network and GO enrichment analysis to determine the core biological function altered after CD147 antibody injection. The CD147 monoclonal antibody suppresses Ang II-induced AAA formation in apoE-/- mice and reduced aortic expansion, elastic lamina degradation, and inflammatory cells accumulation. Bioinformatics analysis showed that Ptk6, Itch, Casp3, and Oas1a were the hub DEPs. These DEPs in the two group were mainly involved in collagen fibril organization, extracellular matrix organization, and muscle contraction. These data robustly demonstrated that CD147 monoclonal antibody suppresses Ang II-induced AAA formation through reduction of inflammatory response and regulation of the above defined hub proteins and biological processes. Thus, the CD147 monoclonal antibody might be a promising target in the treatment of abdominal aortic aneurysm.

Impact of Adipose-Derived Stem Cells on Aortic Tensile Strength in an Abdominal Aortic Aneurysm Model

INTRODUCTION: Abdominal aortic aneurysm (AAA) is a highly morbid condition with treatment currently limited to operative interventions. Prior literature has demonstrated the ability of mesenchymal stem cells (MSCs) in attenuating AAA formation. We aim to demonstrate the mechanical and structural characteristics of the aorta with MSC treatment. METHODS: 16 Yorkshire pigs underwent retroperitoneal exposure of the aorta. Peri-adventitial elastase and collagenase was applied to induce AAA formation. The aorta was treated with media-soaked (aneurysm) or MSC-soaked (treatment) GelFoam. Aortic diameter was monitored serially at postoperative (POD) 0, 7, 14, and 21. Histopathologic staining was performed for hematoxylin and eosin. Tensile strength was measured using a tensiometer and calculations were made for Young’s modulus and peak maximum strain. RESULTS: All animals survived the surgery and postoperative course. At POD21, mean aortic diameter increased to 1.483 cm +/- 0.31 for the aneurysm group vs 1.11 cm +/- 0.04 for the treatment animals (p < 0.004). The elastic modulus for the aneurysm group was 15.83 mPa +/- 1.61 compared to 22.13 mPa +/- 2.34 for the treatment groups (p = 0.034) (Fig. 1). There was no difference in the peak stress (p = 0.564). Medial thickness in the aneurysm group was reduced compared to the treatment animals (590.5 υm +/- 19.35 vs 646.10 υm +/- 11.97, p = 0.044). CONCLUSION: This is the first study to demonstrate the mechanical effects of stem-cell therapy on a porcine AAA model. Young’s modulus characterizes the capacity of tissue to withstand stress and was preserved in the MSC-treated animals. This mechanical test can be utilized to develop additional cell and drug-based therapies for AAA.

Lack of PI 3-kinase isoform p110alpha impairs SMC differentiation and proliferation and promotes aortic aneurysm formation

Abstract Background Proliferation and phenotypic modulation of vascular smooth muscle cells (SMCs) significantly contribute to the functionality of the aortic wall. Dysregulation of underlying signal transduction pathways impairs the vessel wall structure and promote the development and progression of abdominal aortic aneurysms (AAA). The PI 3-kinase (PI3K) isoform p110α is activated downstream of receptor tyrosine kinases (RTKs) and represents the most relevant PI3K isoform in SMCs. Aim This project follows the hypothesis that p110α deficiency impairs proliferation and phenotypic modulation of SMCs as well as the structure of the extracellular matrix (ECM) and therefore promotes the development and progression of AAA. It was investigated how p110α deficiency affects the plasticity of SMCs, the production and structure of ECM components, and the formation of AAA. Methods and results Western blot analyses showed that SMCs isolated from smooth muscle specific p110α−/− (sm-p110α−/−) mice were characterized by decreased expression of the differentiation markers sm-α-actin, calponin and sm-MHC. Mechanistically, phosphorylation of key modulators of the SMC phenotype – AKT1, AKT2, FOXO1, -3 and -4 as well as GSK3β – was impaired in p110α−/− SMCs after RTK stimulation. These findings indicate that phenotypic modulation of p110α−/− SMCs is restricted. In addition, protein expression of elastin and fibrillin was reduced in p110α−/− SMCs. In silico analysis (MatLab macro CT-FIRE and Curvalign) of the ECM produced by SMCs in vitro revealed a significantly reduced elastin fiber length and width in p110α−/− SMCs compared to fibers produced by WT SMCs (p&amp;lt;0.05). Consistently, aortas from sm-p110α−/− mice showed a significantly higher number of elastic fiber breaks specifically in the thoracic section than WT controls (289±31 mmm–2 versus 190±9 mmm–2, n=5, p=0.015). Aortic aneurysms in sm-p110α−/− mice and wild-type littermates were analyzed using the established porcine pancreatic elastase (PPE) model. PPE was perfused into the infrarenal aorta to induce AAA formation. Ultrasound examination of the aorta revealed an enlarged aortic diameter in all PPE-treated mice. However, the increase in aortic diameter in sm-p110α−/− mice (70.16±10.82% mm, n=9) was significant more pronounced compared to wild-type animals (42.44±5.99%, n=10) (p&amp;lt;0.05). Three days after PPE perfusion, the number of elastic fiber breaks was significantly increased, and amount of proliferating SMCs were decreased in the infrarenal aorta of sm-p110α−/− mice compared to WT controls. Conclusion p110α deficiency in SMCs impairs aortic wall structure and promotes the development and progression of aortic aneurysms. Mechanistically, p110α activity maintains a differentiated SMC phenotype as well as the expression and assembly of ECM components. These data identify p110α signaling as a modifiable target for preventive and therapeutic strategies for aortic aneurysms. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft

Abstract 298: Ketosis Prevents Abdominal Aortic Aneurysm Progression And Rupture In Male Rats

Abdominal aortic aneurysms (AAA) are common in aging populations, and its rupture is associated with high mortality. There is no effective medical therapy for the prevention of AAA expansion and rupture. Ketone bodies (KB) are argued to produce novel responses against injury. Serum KB are elevated through a ketogenic diet (KD) or the intake of exogenous KB (EKB). We hypothesize that ketosis can reduce AAA expansion and risk of rupture. Male Sprague-Dawley rats underwent intraluminal exposure to porcine pancreatic elastase (PPE) to induce AAA formation. Rats were also administered daily β-aminopropionitrile (BAPN) to promote AAA rupture. Control rats (N=12) were fed a standard diet (SD). Treated rats were given either a KD (N=9) or EKB (N=10) starting 3 days post AAA induction. Ketosis was verified by blood BHB level &gt;0.5mmol/L. In vivo aortic diameter was evaluated by USG. After 2 weeks, survival animals were harvested, and AAA tissue analysis was performed. Rupture AAA was determined by necropsy. Rats treated with KD consistently remained in ketosis, while rats treated with EKB remained in ketosis for only 8-hour per day. AAA size was significantly reduced (p&lt;0.01 and p&lt;0.05) in both, KD and EKB groups at week 1 and 2 respectively. Rupture rate was also reduced in the KD and EKB groups (22% and 40%, respectively) compared to 67% in the SD group (p=0.03 and 0.12, respectively). AAA trichrome staining demonstrated greater content of collagen in KD and EKB groups when compared to SD group (p=0.08 and 0.02, respectively). AAA gelatin zymography demonstrated significantly decreased active MMP 9 levels in KD and EKB groups (p&lt;0.05) while total MMP 2 was attenuated in the KD group (p&lt;0.01). Ketosis appears to reduce AAA expansion and risk of rupture. This may have resulted from AAA increased collagen deposition as well as decreased MMP activity. These findings provide initial impetus to investigate whether ketosis in patients with small AAAs can prevent expansion and risk of rupture.

Kunming mouse strain is less susceptible to elastase-induced abdominal aortic aneurysms.

BackgroundPorcine pancreatic elastase (PPE) is successfully used to induce abdominal aortic aneurysm (AAA) in mice. However, differences between mouse strains in susceptibility to PPE induction have been reported. Kunming mouse is one of the most frequently used strains in China but whether it is suitable for induction of AAA by PPE application remains unclear.MethodsPPE infusion (1.5 units/ml) in temporary controlled aorta was performed to induce AAAs in both C57BL/6J and Kunming mice. Phosphate‐buffered saline (PBS) application was used as vehicle control. The aorta diameters of all mice were measured at days 0 and 14 after surgery to evaluate the AAA formation.ResultsAfter 14 days of PPE or PBS infusion, all mice were sacrificed and aorta tissues were collected for histological staining analysis. At the 14th day after infusion, PPE successfully induced aortic dilation in Kunming mice and typical AAA in C57BL/6J mice. The aorta diameter increased by 0.23 mm in Kunming mice after PPE infusion, while it was 0.72 mm in the C57BL/6J strain. PPE induced mild elastin degradation, smooth muscle cell (SMC) depletion and mural leucocyte infiltration in Kunming mice, but in PPE‐sensitive C57BL/6J mice, it induced total loss of SMCs, elastin disappearance and diffused infiltrated leucocytes in aortic aneurysmal segments. The effects of PPE in inducing angiogenesis and upregulating matrix metalloproteinase 2 and 9 expression in Kunming mice were also weaker than that in C57BL/6J mice.ConclusionAt the reported dose of PPE, Kunming mouse is not as susceptible to AAA formation as C57BL/6J mice. The failure of PPE to induce AAA formation in Kunming mice may be associated to its inability to boost a strong inflammatory response.

Circular RNA Cdyl promotes abdominal aortic aneurysm formation by inducing M1 macrophage polarization and M1-type inflammation

Macrophage polarization plays a crucial role in regulating abdominal aortic aneurysm (AAA) formation. Circular RNAs (circRNAs) are important regulators of macrophage polarization during the development of cardiovascular diseases. How-ever, the roles of circRNAs in regulating AAA formation through modulation of macrophage polarization remain unknown. In the present study, we compared circRNA microarray data under two distinct polarizing conditions (M1 and M2 macrophages) and identified an M1-enriched circRNA, circCdyl. Loss- and gain-of-function assay results demonstrated that circCdyl overexpression accelerated angiotensin II (Ang II)- and calcium chloride (CaCl2)-induced AAA formation by promoting M1 polarization and M1-type inflammation, while circCdyl deficiency showed the opposite effects. RNA pulldown, mass spectrometry analysis, and RNA immunoprecipitation (RIP) assays were conducted to elucidate the underlying mechanisms by which circCdyl regulates AAA formation and showed that circCdyl promotes vascular inflammation and M1 polarization by inhibiting interferon regulatory factor 4 (IRF4) entry into the nucleus, significantly inducing AAA formation. In addition, circCdyl was shown to act as a let-7c sponge, promoting C/EBP-δ expression in macrophages to induce M1 polarization. Our results indicate an important role for circCdyl-mediated macrophage polarization in AAA formation and provide a potent therapeutic target for AAA treatment.

Abstract MP23: Knockout Of Pgc1α In Endothelial Cells Augments Abdominal Aortic Aneurysm Formation

Introduction: It is well described that changes in the aortic media are crucial in the development of Abdominal Aortic Aneurysms (AAA), however the involvement of the endothelium is not as clear. The endothelium is the first vascular layer exposed to blood flow and aneurysms preferentially form in areas of disturbed flow. The regulatory mechanisms as to why aneurysms form at these sites remains unclear. Here we propose that the transcription coactivator peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) plays a pivotal role in the endothelium’s response to AAA formation in a shear responsive manner. Methods: 10-12 week old male mice deficient in endothelial specific PGC1α (KO) and their corresponding wild type background mice (WT) were treated with 0.75 mg/kg/day of Ang II. Our primary endpoints were measurement of the maximal width of the suprarenal aortas, frequency, and complexity based on the Daugherty classification. To determine the effects of shear, HAECs exposed to steady flow (15 dyn/cm 2 ) and disturbed flow (0±6 dyn/cm 2 at 1 Hz) for 12 hours were compared to determine if mRNA expression were different for PGC1α and SIRT1. Results: AAA incidence and severity were increased in ANG II-infused KO compared to the WT mice (100% vs. 20%, respectively). Measurements of excised aortas showed that KO mice exhibited larger AAA fold-increase compared to WT (2.00 ± 0.11 fold increase vs. 0.43 ± 0.19 fold increase, p-value 0.0002). The KO male mice also exhibited a more complex phenotype. HAECs that underwent disturbed flow had a significant decrease in expression of SIRT1 (p = 0.02) and trended towards a decrease in expression of PGC1α (p = 0.06). Conclusions: Our data strongly suggests that knockout of endothelial specific PGC1α leads to more frequent, complex and larger AAAs primarily in the suprarenal aorta, which is an area of disturbed flow. Interestingly, the addition of ANG II alone was able to induce AAA formation in our model. Additionally, we show that PGC1α and SIRT1 are regulated in a shear responsive manner. Taken together, endothelial PGC1α likely plays a regulatory role in AAA formation through shear-related mechanisms.

Targeting Tyrosine Hydroxylase for Abdominal Aortic Aneurysm: Impact on Inflammation, Oxidative Stress, and Vascular Remodeling.

[Figure: see text].

MKL1 cooperates with p38MAPK to promote vascular senescence, inflammation, and abdominal aortic aneurysm

Abdominal aortic aneurysm (AAA) is a catastrophic disease with little effective therapy. Myocardin related transcription factor A (MRTFA, MKL1) is a multifaceted transcription factor, regulating diverse biological processes. However, a detailed understanding of the mechanistic role of MKL1 in AAA has yet to be elucidated. In this study, we showed induced MKL1 expression in thoracic and abdominal aneurysmal tissues, respectively in both mice and humans. MKL1 global knockout mice displayed reduced AAA formation and aortic rupture compared with wild-type mice. Both gene deletion and pharmacological inhibition of MKL1 markedly protected mice from aortic dissection, an early event in Angiotensin II (Ang II)-induced AAA formation. Loss of MKL1 was accompanied by reduced senescence/proinflammation in the vessel wall and cultured vascular smooth muscle cells (VSMCs). Mechanistically, a deficiency in MKL1 abolished AAA-induced p38 mitogen activated protein kinase (p38MAPK) activity. Similar to MKL1, loss of MAPK14 (p38α), the dominant isoform of p38MAPK family in VSMCs suppressed Ang II-induced AAA formation, vascular inflammation, and senescence marker expression. These results reveal a molecular pathway of AAA formation involving MKL1/p38MAPK stimulation and a VSMC senescent/proinflammatory phenotype. These data support targeting MKL1/p38MAPK pathway as a potential effective treatment for AAA.

Nanoparticle-Mediated Delivery of Pitavastatin to Monocytes/Macrophages Inhibits Angiotensin II-Induced Abdominal Aortic Aneurysm Formation in Apoe-/- Mice.

Aim: Abdominal aortic aneurysm (AAA) is a lethal and multifactorial disease. To prevent a rupture and dissection of enlarged AAA, prophylactic surgery and stenting are currently available. There are, however, no medical therapies preventing these complications of AAA. Statin is one of the candidates, but its efficacy on AAA formation/progression remains controversial. We have previously demonstrated that nanoparticles (NPs) incorporating pitavastatin (Pitava-NPs)—clinical trials using these nanoparticles have been already conducted—suppressed progression of atherosclerosis in apolipoprotein E-deficient ( Apoe −/− ) mice. Therefore, we have tested a hypothesis that monocytes/macrophages-targeting delivery of pitavastatin prevents the progression of AAA. Methods: Angiotensin II was intraperitoneally injected by osmotic mini-pumps to induce AAA formation in Apoe −/− mice. NPs consisting of poly(lactic-co-glycolic acid) were used for in vivo delivery of pitavastatin to monocytes/macrophages. Results: Intravenously administered Pitava-NPs (containing 0.012 mg/kg/week pitavastatin) inhibited AAA formation accompanied with reduction of macrophage accumulation and monocyte chemoattractant protein-1 (MCP-1) expression. Ex vivo molecular imaging revealed that Pitava-NPs not only reduced macrophage accumulation but also attenuated matrix metalloproteinase activity in the abdominal aorta, which was underpinned by attenuated elastin degradation. Conclusion: These results suggest that Pitava-NPs inhibit AAA formation associated with reduced macrophage accumulation and MCP-1 expression. This clinically feasible nanomedicine could be an innovative therapeutic strategy that prevents devastating complications of AAA.

Salvia miltiorrhiza-Derived Sal-miR-58 Induces Autophagy and Attenuates Inflammation in Vascular Smooth Muscle Cells

Autophagy is associated with the cytoprotection of physiological processes against inflammation and oxidative stress. Salvia miltiorrhiza possesses cardiovascular protective actions and has powerful anti-oxidative and anti-inflammatory effects; however, whether and how Salvia miltiorrhiza-derived microRNAs (miRNAs) protect vascular smooth muscle cells (VSMCs) by inducing autophagy across species are unknown. We first screened and identified Sal-miR-58 from Salvia miltiorrhiza as a natural autophagy inducer. Synthetic Sal-miR-58 suppresses chronic angiotensin II (Ang II) infusion-induced abdominal aortic aneurysm (AAA) formation in mice, as well as induces autophagy in VSMCs and attenuates the inflammatory response elicited by Ang II in vivo and in vitro. Mechanistically, Sal-miR-58 downregulates Krüppel-like factor 3 (KLF3) expression through direct binding to the 3′ UTR of KLF3, which in turn relieves KLF3 repression of E3 ubiquitin ligase neural precursor cell-expressed developmentally downregulated 4-like (NEDD4L) expression, whereas NEDD4L upregulation increases the ubiquitination and degradation of the platelet isoform of phosphofructokinase (PFKP), subsequently leading to a decrease in the activation of Akt/mammalian target of rapamycin (mTOR) signaling and facilitating VSMC autophagy induced by Sal-miR-58 in the context of chronic Ang II stimulation and aneurysm formation. Our results provide the first evidence that plant-derived Sal-miR-58 induces autophagy and attenuates inflammation in VSMCs through cross-species modulation of the KLF3/NEDD4L/PFKP regulatory pathway.

MiR-126a-5p limits the formation of abdominal aortic aneurysm in mice and decreases ADAMTS-4 expression.

Abdominal aortic aneurysm (AAA) is a serious vascular disease featured by inflammatory infiltration in aortic wall, aortic dilatation and extracellular matrix (ECM) degradation. Dysregulation of microRNAs (miRNAs) is implicated in AAA progress. By profiling miRNA expression in mouse AAA tissues and control aortas, we noted that miR‐126a‐5p was down‐regulated by 18‐fold in AAA samples, which was further validated with real‐time qPCR. This study was performed to investigate miR‐126a‐5p's role in AAA formation. In vivo, a 28‐d infusion of 1 μg/kg/min Angiotensin (Ang) II was used to induce AAA formation in Apoe‐/‐ mice. MiR‐126a‐5p (20 mg/kg; MIMAT0000137) or negative control (NC) agomirs were intravenously injected to mice on days 0, 7, 14 and 21 post‐Ang II infusion. Our data showed that miR‐126a‐5p overexpression significantly improved the survival and reduced aortic dilatation in Ang II‐infused mice. Elastic fragment and ECM degradation induced by Ang II were also ameliorated by miR‐126a‐5p. A strong up‐regulation of ADAM metallopeptidase with thrombospondin type 1 motif 4 (ADAMTS‐4), a secreted proteinase that regulates matrix degradation, was observed in smooth muscle cells (SMCs) of aortic tunica media, which was inhibited by miR‐126a‐5p. Dual‐luciferase results demonstrated ADAMTS‐4 as a new and valid target for miR‐126a‐5p. In vitro, human aortic SMCs (hASMCs) were stimulated by Ang II. Gain‐ and loss‐of‐function experiments further confirmed that miR‐126‐5p prevented Ang II‐induced ECM degradation, and reduced ADAMTS‐4 expression in hASMCs. In summary, our work demonstrates that miR‐126a‐5p limits experimental AAA formation and reduces ADAMTS‐4 expression in abdominal aortas.

Excessive EP4 Signaling in Smooth Muscle Cells Induces Abdominal Aortic Aneurysm by Amplifying Inflammation.

Excessive prostaglandin E2 production is a hallmark of abdominal aortic aneurysm (AAA). Enhanced expression of prostaglandin E2 receptor EP4 (prostaglandin E receptor 4) in vascular smooth muscle cells (VSMCs) has been demonstrated in human AAAs. Although moderate expression of EP4 contributes to vascular homeostasis, the roles of excessive EP4 in vascular pathology remain uncertain. We aimed to investigate whether EP4 overexpression in VSMCs exacerbates AAAs. Approach and Results: We constructed mice with EP4 overexpressed selectively in VSMCs under an SM22α promoter (EP4-Tg). Most EP4-Tg mice died within 2 weeks of Ang II (angiotensin II) infusion due to AAA, while nontransgenic mice given Ang II displayed no overt phenotype. EP4-Tg developed much larger AAAs than nontransgenic mice after periaortic CaCl2 application. In contrast, EP4fl/+;SM22-Cre;ApoE-/- and EP4fl/+;SM22-Cre mice, which are EP4 heterozygous knockout in VSMCs, rarely exhibited AAA after Ang II or CaCl2 treatment, respectively. In Ang II-infused EP4-Tg aorta, Ly6Chi inflammatory monocyte/macrophage infiltration and MMP-9 (matrix metalloprotease-9) activation were enhanced. An unbiased analysis revealed that EP4 stimulation positively regulated the genes binding cytokine receptors in VSMCs, in which IL (interleukin)-6 was the most strongly upregulated. In VSMCs of EP4-Tg and human AAAs, EP4 stimulation caused marked IL-6 production via TAK1 (transforming growth factor-β-activated kinase 1), NF-κB (nuclear factor-kappa B), JNK (c-Jun N-terminal kinase), and p38. Inhibition of IL-6 prevented Ang II-induced AAA formation in EP4-Tg. In addition, EP4 stimulation decreased elastin/collagen cross-linking protein LOX (lysyl oxidase) in both human and mouse VSMCs. Dysregulated EP4 overexpression in VSMCs promotes inflammatory monocyte/macrophage infiltration and attenuates elastin/collagen fiber formation, leading to AAA exacerbation.

P1937Deficiency of PI 3-kinase isoform p110alpha in smooth muscle cells impairs vascular integrity and promotes aortic aneurysm formation

Abstract Background The pathobiology of aortic aneurysms is characterized by vascular inflammation, extracellular matrix degeneration, and particularly by loss and dedifferentiation of vascular smooth muscle cells (SMCs). In SMCs, the PI 3-kinase isoform p110α mediates receptor tyrosine kinase dependent proliferation, chemotaxis, and survival. Smooth muscle specific p110α deficient mice (SM-p110α−/− mice) display reduced medial wall thickness, substantially reduced neointima formation and media hypertrophy after balloon injury of the carotid artery. Objective We hypothesized that loss of p110α signaling impairs vascular integrity and promotes development and progression of abdominal aortic aneurysms (AAA). We aimed to elucidate the impact of p110α deficiency on vascular integrity, SMC phenotypic modulation, vascular inflammation, and AAA formation. Methods and results Ultra-structural characterization of aortic wall morphology in abdominal aortas from SM-p110α−/− mice by transmission electron microscopy (TEM) revealed disarranged structure of tunica media as indicated by disorganized elastic fibers, detached SMCs, and elastic fiber breaks. Western blots showed reduced elastin and fibrillin expression in SMCs from p110α−/− mice. Media thickness was significantly reduced in abdominal aortas from SM-p110α−/− mice compared to wild type (WT) controls (29.0±3.1 vs. 42.5±4.1 μm). Lack of p110α decreased expression of differentiation markers SM-α-actin and SM-MHC. p110α deficiency significantly diminished responsiveness of aortic rings to vasodilator acetylcholine. These data indicate loss of differentiation and impaired contractility of p110α−/− SMCs. We subjected SM-p110α−/− mice and WT littermate controls to the porcine pancreatic elastase (PPE) model of AAA. PPE was infused into the infrarenal aorta, respectively, to induce AAA formation. Ultrasonic examination of abdominal aortas demonstrated an enlarged aortic diameter in PPE challenged mice. AAA formation was significantly (p&lt;0.01) enhanced in SM-p110α−/− (0.46±0.12 mm, n=8) compared to SM-p110α+/+ mice (0.18±0.03 mm, n=4). These data indicate a protective function of p110α in AAA formation. Immunocytochemistry of the aortic medial compartment from PPE-perfused SM-p110α−/− mice revealed significantly increased MOMA-2+ monocyte/macrophage content indicating augmented aortic inflammation during AAA formation compared to WT controls. Furthermore, SMCs from SM-p110α−/− mice expressed reduced amounts of anti-inflammatory angiopoietin1 compared to p110α+/+ SMCs. Moreover, frequent apoptotic/necrotic SMCs were found in the aortic media of SM-p110α−/− mice by TEM, potentially contributing to vascular inflammation in a critical fashion. Conclusion These data indicate that p110α signaling critically contributes to vascular integrity via maintaining SMC plasticity, elastic fiber homeostasis, and anti-inflammatory processes. Consequently, lack of proper p110α signaling promotes progression of AAA formation.

Copper Transporter ATP7A (Copper-Transporting P-Type ATPase/Menkes ATPase) Limits Vascular Inflammation and Aortic Aneurysm Development: Role of MicroRNA-125b.

Copper (Cu) is essential micronutrient, and its dysregulation is implicated in aortic aneurysm (AA) development. The Cu exporter ATP7A (copper-transporting P-type ATPase/Menkes ATPase) delivers Cu via the Cu chaperone Atox1 (antioxidant 1) to secretory Cu enzymes, such as lysyl oxidase, and excludes excess Cu. Lysyl oxidase is shown to protect against AA formation. However, the role and mechanism of ATP7A in AA pathogenesis remain unknown. Approach and Results: Here, we show that Cu chelator markedly inhibited Ang II (angiotensin II)-induced abdominal AA (AAA) in which ATP7A expression was markedly downregulated. Transgenic ATP7A overexpression prevented Ang II-induced AAA formation. Conversely, Cu transport dysfunctional ATP7Amut/+/ApoE-/- mice exhibited robust AAA formation and dissection, excess aortic Cu accumulation as assessed by X-ray fluorescence microscopy, and reduced lysyl oxidase activity. In contrast, AAA formation was not observed in Atox1-/-/ApoE-/- mice, suggesting that decreased lysyl oxidase activity, which depends on both ATP7A and Atox1, was not sufficient to develop AAA. Bone marrow transplantation suggested importance of ATP7A in vascular cells, not bone marrow cells, in AAA development. MicroRNA (miR) array identified miR-125b as a highly upregulated miR in AAA from ATP7Amut/+/ApoE-/- mice. Furthermore, miR-125b target genes (histone methyltransferase Suv39h1 and the NF-κB negative regulator TNFAIP3 [tumor necrosis factor alpha induced protein 3]) were downregulated, which resulted in increased proinflammatory cytokine expression, aortic macrophage recruitment, MMP (matrix metalloproteinase)-2/9 activity, elastin fragmentation, and vascular smooth muscle cell loss in ATP7Amut/+/ApoE-/- mice and reversed by locked nucleic acid-anti-miR-125b infusion. ATP7A downregulation/dysfunction promotes AAA formation via upregulating miR-125b, which augments proinflammatory signaling in a Cu-dependent manner. Thus, ATP7A is a potential therapeutic target for inflammatory vascular disease.

Apelin protects against abdominal aortic aneurysm and the therapeutic role of neutral endopeptidase resistant apelin analogs.

Abdominal aortic aneurysm (AAA) remains the second most frequent vascular disease with high mortality but has no approved medical therapy. We investigated the direct role of apelin (APLN) in AAA and identified a unique approach to enhance APLN action as a therapeutic intervention for this disease. Loss of APLN potentiated angiotensin II (Ang II)-induced AAA formation, aortic rupture, and reduced survival. Formation of AAA was driven by increased smooth muscle cell (SMC) apoptosis and oxidative stress in Apln-/y aorta and in APLN-deficient cultured murine and human aortic SMCs. Ang II-induced myogenic response and hypertension were greater in Apln-/y mice, however, an equivalent hypertension induced by phenylephrine, an α-adrenergic agonist, did not cause AAA or rupture in Apln-/y mice. We further identified Ang converting enzyme 2 (ACE2), the major negative regulator of the renin-Ang system (RAS), as an important target of APLN action in the vasculature. Using a combination of genetic, pharmacological, and modeling approaches, we identified neutral endopeptidase (NEP) that is up-regulated in human AAA tissue as a major enzyme that metabolizes and inactivates APLN-17 peptide. We designed and synthesized a potent APLN-17 analog, APLN-NMeLeu9-A2, that is resistant to NEP cleavage. This stable APLN analog ameliorated Ang II-mediated adverse aortic remodeling and AAA formation in an established model of AAA, high-fat diet (HFD) in Ldlr-/- mice. Our findings define a critical role of APLN in AAA formation through induction of ACE2 and protection of vascular SMCs, whereas stable APLN analogs provide an effective therapy for vascular diseases.

ASB17061, a novel chymase inhibitor, prevented the development of angiotensin II-induced abdominal aortic aneurysm in apolipoprotein E-deficient mice

Our aim was to examine the effects of ASB17061, an orally active novel chymase inhibitor, on angiotensin II–induced abdominal aortic aneurysm (AAA) in apolipoprotein E–deficient mice. Oral administration of ASB17061 (10 mg/kg) significantly suppressed angiotensin II–induced AAA formation in these mice. The pro-matrix metalloproteinase-9 (pro-MMP-9) level in AAA lesions was significantly suppressed by ASB17061 treatment, indicating that ASB17061 inhibited the accumulation of pro-MMP-9–producing cells in AAA lesions. Mouse mast cell protease 4 (mMCP-4, human chymase ortholog) was injected into BALB/c mice intraperitoneally to examine the ability of mMCP-4 to induce the accumulation of pro-MMP-9–producing cells. An intraperitoneal injection of mMCP-4 induced the accumulation of pro-MMP-9–producing cells including CD11b + Gr-1 + cells. Taken together, these data indicate that ASB17061 is a promising novel oral therapeutic agent for human AAA.

LncRNA H19 promotes vascular inflammation and abdominal aortic aneurysm formation by functioning as a competing endogenous RNA

Abdominal aortic aneurysm (AAA) is accepted as a chronic vascular inflammatory disease. However, how the inflammatory response is regulated during AAA formation is not fully understood. This study was undertaken to determine whether the long noncoding RNA (lncRNA) H19 (H19) promotes AAA formation by enhancing aortic inflammation. qRT-PCR detected the upregulation of H19 in human and mouse AAA tissue samples. Co-staining for H19 and the macrophage marker MAC-2 showed that H19 was located in vascular smooth muscle cells (VSMCs) and infiltrating aortic macrophages. In vivo overexpression of H19 increased vascular inflammation and induced AAA formation, which was supported by exacerbated aortic morphology, maximum aortic diameter values, elastin degradation, expression of interleukin-6 (IL-6) and macrophage chemoattractant protein-1 (MCP-1), and macrophage infiltration. H19 suppression resulted in the opposite effects. A rescue experiment indicated that IL-6 neutralization significantly mitigated the aortic inflammation and AAA formation evoked by H19 overexpression. Luciferase reporter assays and ex vivo experiments using VSMCs and macrophages confirmed that H19 induced aneurysm formation in part via endogenous competition with the let-7a microRNA to induce the transcription of its target gene, IL-6. This mechanism was further validated by in vivo experiments using a mutant H19 that could not effectively bind let-7a. Collectively, our study revealed a pathogenic H19/let-7a/IL-6 inflammatory pathway in AAA formation, which offers a new potential therapeutic strategy for AAA.

Caloric Restriction Exacerbates Angiotensin II-Induced Abdominal Aortic Aneurysm in the Absence of p53.

p53-dependent vascular smooth muscle cell senescence is a key pathological process of abdominal aortic aneurysm (AAA). Caloric restriction (CR) is a nonpharmacological intervention that prevents AAA formation. However, whether p53 is indispensable to the protective role of CR remains unknown. In this study, we investigated the necessity of p53 in the beneficial role of CR in AAA formation and the underlying mechanisms. We subjected p53+/+ and p53-/- mice to 12 weeks of CR and then examined the incidence of Ang II (angiotensin II)-induced AAA formation. We found that both CR and p53 knockout reduced Ang II-induced AAA formation; however, CR markedly increased the incidence of AAA formation and exacerbated aortic elastin degradation in p53-/- mice, accompanied by increased vascular senescence, reactive oxygen species generation, and reduced energy production. Analysis of mitochondrial respiratory activity revealed that dysfunctional complex IV accounts for the abnormal mitochondrial respiration in p53-/- vascular smooth muscle cells treated by CR serum. Mechanistically, ablation of p53 almost totally blocked the protective role of CR by inhibiting SCO2 (cytochrome C oxidase assembly protein 2)-dependent mitochondrial complex IV activity. Overexpression of SCO2 restored the beneficial effect of CR on antagonizing Ang II-induced expression of AAA-related molecules and reactive oxygen species generation in p53-/- vascular smooth muscle cells. Together, our findings demonstrate that the existence of p53 in vascular smooth muscle cells is critical to the protective role of CR in Ang II-induced AAA formation by maintaining an appropriate mitochondrial function.