Experimental Abdominal Aortic Aneurysm Research Articles

Decreased atherosclerosis and abdominal aortic aneurysm in mice deficient in polymeric immunoglobulin receptor

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): La Caixa Banking Foundation and Comunidad de Madrid Background Polymeric immunoglobulin receptor (PIGR) is a transmembrane protein involved in the transcytosis of polymeric immunoglobulins that is widely expressed in mucosal epithelial cells. Recent data from our group described increased PIGR levels in plasma and tissue of atherosclerotic subjects. However, the role for PIGR in the mechanisms involved in vascular remodeling has not been addressed. Purpose To analyse the role for PIGR in experimental mouse models of atherosclerosis and abdominal aortic aneurysm (AAA). Methods We analyzed PIGR expression, secretion and distribution in early atherosclerotic plaques and AAA tissues compared to healthy aortic samples, as well as in macrophages derived from the human monocytic THP-1 cell line. Next, we studied the effect of global PIGR deficiency in experimental atherosclerosis (Ldlr-/- Pigr-/- mice fed with atherogenic diet for 10 weeks). In addition, the contribution of hematopoietic PIGR was assessed in both experimental atherosclerosis and AAA (Ldlr-/- mice fed with atherogenic diet and 1 µg/Kg/min angiotensin II infusion for 28 days) by bone marrow transplantation experiments. Finally, bone marrow-derived macrophages (BMDMs) from Pigr-/- and control mice were isolated to assess their inflammatory expression profile and modulation of polarization in response to oxidized LDL (ox-LDL), as well as their ability to promote foam cell formation. Results PIGR expression was increased in the intima of early human atherosclerotic lesions and showed colocalization with CD68+ expression. PIGR expression was also increased during the in vitro differentiation of THP-1 monocytes to macrophages with phorbol myristate acetate. Both global and hematopoietic-specific PIGR deficient mice displayed reduced plaque size in the aortic sinuses as well as a strong decrease in macrophage infiltration (CD68+ cells), while no changes in smooth muscle cells (α-SMA+ cells) and collagen content were observed. Regarding AAA, increased PIGR levels were found in human AAA tissues, associated with infiltrating CD68+ cells. PIGR deficiency in hematopoietic cells also showed significantly reduced AAA dilatation and reduced macrophage infiltration. In vitro experiments with BMDMs obtained from Pigr-/- mice and stimulated with ox-LDL showed reduced proinflammatory gene expression (TNF-a and IL-6) and M1 markers (iNOS and CD11c) as well as diminished foam cell formation. Conclusions PIGR deficiency in hematopoietic cells decreases atherosclerosis and AAA progression by modulating macrophage inflammatory responses, suggesting a novel proinflammatory role for PIGR in vascular pathologies. Future experiments will test the potential therapeutic effect of modulating PIGR to dampen the inflammatory profile in vascular diseases.Results in human and mouse studies

Colchicine Blocks Abdominal Aortic Aneurysm Development by Maintaining Vascular Smooth Muscle Cell Homeostasis.

Development of non-surgical treatment of human abdominal aortic aneurysm (AAA) has clinical significance. Colchicine emerges as an effective therapeutic regimen in cardiovascular diseases. Yet, whether colchicine slows AAA growth remain controversy. Here, we demonstrated that daily intragastric administration of low-dose colchicine blocked AAA formation, prevented vascular smooth muscle cell (SMC) phenotype switching and apoptosis, and vascular inflammation in both peri-aortic CaPO4 injury and subcutaneous angiotensin-II infusion induced experimental AAA mice models. Mechanistically, colchicine increased global mRNA stability by inhibiting the METTL14/YTHDC1-mediated m6A modification, resulting in increased sclerostin (SOST) expression and consequent inactivation of the WNT/β-catenin signaling pathway in vascular SMCs from mouse AAA lesions and in cultured human aortic SMCs. Moreover, human and mouse AAA lesions all showed increased m6A methylation, decreased SOST expression, and skewed synthetic SMC de-differentiation phenotype, compared to those without AAA. This study uncovers a novel mechanism of colchicine in slowing AAA development by using the METTL14/SOST/WNT/β-catenin axis to control vascular SMC homeostasis in mouse aortic vessels and in human aortic SMCs. Therefore, use of colchicine may benefit AAA patients in clinical practice.

The incidence rate and histological characteristics of intimal hyperplasia in elastase-induced experimental abdominal aortic aneurysms in mice.

Intimal hyperplasia (IH) is a negative vascular remodeling after arterial injury. IH occasionally occurs in elastase-induced abdominal aortic aneurysm (AAA) mouse models. This study aims to clarify the incidence and histological characteristics of IH in aneurysmal mice. A retrospective study was conducted by including 42 male elastase-induced mouse AAA models. The IH incidence, aortic diameters with or without IH, and hyperplasia lesional features of mice were analyzed. Among 42 elastase-induced AAA mouse models, 10 mice developed mild IH (24%) and severe IH was found in only 2 mice (5%). The outer diameters of the AAA segments in mice with and without IH did not show significant difference. Both mild and severe IH lesions show strong smooth muscle cell positive staining, but endothelial cells were occasionally observed in severe IH lesions. There was obvious macrophage infiltration in the IH lesions of the AAA mouse models, especially in mice with severe IH. However, only a lower numbers of T cells and B cells were found in the IH lesion. Local cell-secreted matrix metalloproteinases (MMP) 2 was highly expressed in all IH lesions, but MMP9 was only overexpressed in severe lesions. In conclusion, this study is the first to demonstrate the occurrence of aneurysmal IH and its histological characteristics in an elastase-induced mouse AAA model. This will help researchers better understand this model, and optimize it for use in AAA-related research.

Crosstalk of platelets with macrophages and fibroblasts aggravates inflammation, aortic wall stiffening, and osteopontin release in abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) is a highly lethal disease with progressive dilatation of the abdominal aorta accompanied by degradation and remodelling of the vessel wall due to chronic inflammation. Platelets play an important role in cardiovascular diseases, but their role in AAA is poorly understood. The present study revealed that platelets play a crucial role in promoting AAA through modulation of inflammation and degradation of the extracellular matrix (ECM). They are responsible for the up-regulation of SPP1 (osteopontin, OPN) gene expression in macrophages and aortic tissue, which triggers inflammation and remodelling and also platelet adhesion and migration into the abdominal aortic wall and the intraluminal thrombus (ILT). Further, enhanced platelet activation and pro-coagulant activity result in elevated gene expression of various cytokines, Mmp9 and Col1a1 in macrophages and Il-6 and Mmp9 in fibroblasts. Enhanced platelet activation and pro-coagulant activity were also detected in AAA patients. Further, we detected platelets and OPN in the vessel wall and in the ILT of patients who underwent open repair of AAA. Platelet depletion in experimental murine AAA reduced inflammation and ECM remodelling, with reduced elastin fragmentation and aortic diameter expansion. Of note, OPN co-localized with platelets, suggesting a potential role of OPN for the recruitment of platelets into the ILT and the aortic wall. In conclusion, our data strongly support the potential relevance of anti-platelet therapy to reduce AAA progression and rupture in AAA patients.

Application and challenges of TCR and BCR sequencing to investigate T- and B-cell clonality in elastase-induced experimental murine abdominal aortic aneurysm.

An abdominal aortic aneurysm (AAA) is a life-threatening cardiovascular disease. Although its pathogenesis is still poorly understood, recent evidence suggests that AAA displays autoimmune disease characteristics. Particularly, T cells responding to AAA-related antigens in the aortic wall may contribute to an initial immune response. Single-cell RNA (scRNA) T cell receptor (TCR) and B cell receptor (BCR) sequencing is a powerful tool for investigating clonality. However, difficulties such as limited numbers of isolated cells must be considered during implementation and data analysis, making biological interpretation challenging. Here, we perform a representative single-cell immune repertoire analysis in experimental murine AAA and show a reliable bioinformatic processing pipeline highlighting opportunities and limitations of this approach. We performed scRNA TCR and BCR sequencing of isolated lymphocytes from the infrarenal aorta of male C57BL/6J mice 3, 7, 14, and 28 days after AAA induction via elastase perfusion of the aorta. Sham-operated mice at days 3 and 28 and non-operated mice served as controls. Comparison of complementarity-determining region (CDR3) length distribution of 179 B cells and 796 T cells revealed neither differences between AAA and control nor between the disease stages. We found no clonal expansion of B cells in AAA. For T cells, we identified several clones in 11 of 16 AAA samples and one of eight control samples. Immune receptor repertoire comparison indicated that only a few clones were shared between the individual AAA samples. The most frequently used V-genes in the TCR beta chain in AAA were TRBV3, TRBV19, and the splicing variant TRBV12-2 + TRBV13-2. We found no clonal expansion of B cells but evidence for clonal expansion of T cells in elastase-induced AAA in mice. Our findings imply that a more precise characterization of TCR and BCR distribution requires a more extensive number of lymphocytes to prevent undersampling and potentially detect rare clones. Thus, further experiments are necessary to confirm our findings. In summary, this paper examines TCR and BCR sequencing results, identifies limitations and pitfalls, and offers guidance for future studies.

Olfactory receptor 2 signaling drives experimental abdominal aortic aneurysm formation

Abstract Background Abdominal aortic aneurysms (AAA) are defined as enlargement above 3cm or 50% of its original diameter. Therapeutic options are limited to surgical and endovascular treatment, while no pharmacological interventions are available. Chronic inflammation of the vessel wall and in particularly macrophage infiltration has been associated with disease progression, leading to extensive tissue remodeling, yet the underlying mechanisms are incompletely understood. A recent study has shown extra-nasal expression of the olfactory receptor 2 (OLFR2) on vascular macrophages, in which activation induced a pro-atherogenic response. The role of macrophage OLFR2 signaling in AAA, however, is unknown. Methods & Results To determine whether the OLFR2 ortholog OR6A2 is expressed in human AAA, we performed in silico micro-array analysis of human AAA tissue. OR6A2 was shown to be specifically upregulated in AAA tissue with thrombus formation compared to healthy control tissue. We confirmed OR6A2 expression on macrophages by immunofluorescence. To determine OLFR2 expression among vascular immune cells in mice, we induced AAA formation by porcine pancreas (PPE) infusion into the infrarenal aorta of C57BL6/J (WT) mice and performed spectral cytometry utilizing a 26-marker panel at baseline, day 7 and day 28. OLFR2 expression peaked at day 7 and decreased to baseline levels at day 28 particularly on pro-inflammatory monocytes, mixed resident / migratory-like macrophages and pro-inflammatory macrophages, while OLFR2 was not detectable on tissue resident-like macrophages. To assess a functional role of OLFR2 in AAA formation, we induced AAA in OLFR2-deficient (KO) mice and WT controls by PPE. Ultrasound analysis of the abdominal aorta revealed protection from AAA formation in KO mice. Histological analysis showed significantly reduced macrophage while increased smooth muscle cell content in aortic sections of KO mice compared to WT controls. This was accompanied by higher collagen content in KO mice. Additionally, plasma levels of the pro-inflammatory cytokines TNF-alpha and Interferon-gamma were significantly reduced in KO mice 28 days post PPE. Bulk transcriptome analysis corroborated these findings and indicated increased pathways associated with vascular smooth muscle cell contractility while decreased leukocyte activation in KO aortas. Conclusion We show that OLFR2 deficiency in mice attenuates abdominal aortic aneurysm development, aortic remodeling and pro-inflammatory response. OLFR2 might thus present a novel target for drug-based AAA therapies.

Colchicine Protects Against the Development of Experimental Abdominal Aortic Aneurysm.

Abdominal aortic aneurysm (AAA) is characterized by at least 1.5-fold enlargement of the infrarenal aorta, a ruptured AAA is life-threatening. Colchicine is a medicine used to treat gout and familial Mediterranean fever, and recently it was approved to reduce the risk of cardiovascular events in adult patients with established atherosclerotic disease. With an AAA mice model created by treatment with porcine pancreatic elastase (PPE) and beta aminopropionitrile (BAPN), this work was designed to explore whether colchicine could protect against the development of AAA. Here, we showed that colchicine could limit AAA formation, as evidenced by the decreased total aortic weight per body weight, AAA incidence, maximal abdominal aortic diameter and collagen deposition. We also found that colchicine could prevent the phenotypic switching of vascular smooth muscle cells from a contractile to synthetic state during AAA. In addition, it was demonstrated that colchicine was able to reduce vascular inflammation, oxidative stress, cell pyroptosis and immune cells infiltration to the aortic wall in the AAA mice model. Finally, it was proved that the protective action of colchicine against AAA formation was mainly mediated by preventing immune cells infiltration to the aortic wall. In summary, our findings demonstrated that colchicine could protect against the development of experimental AAA, providing a potential therapeutic strategy for AAA intervention in the clinic.

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.

An evidence update to explore molecular targets and protective mechanisms of apigenin against abdominal aortic aneurysms based on network pharmacology and experimental validation.

Abdominal aortic aneurysms (AAA) is a life-threatening disease and the incidence of AAA is still on the rise in recent years. Numerous studies suggest that dietary moderate consumption of polyphenol exerts beneficial effects on cardiovascular disease. Apigenin (API) is a promising dietary polyphenol and possesses potent beneficial effects on our body. Although our previous study revealed protective effects of API on experimental AAA formation, up till now few studies were carried out to further investigate its involved molecular mechanisms. In the present study, network pharmacology combined molecular docking and experimental validation was used to explore API-related therapeutic targets and mechanisms in the treatment of AAA. Firstly, we collected 202 API-related therapeutic targets and 2475 AAA-related pathogenetic targets. After removing duplicates, a total of 68 potential therapeutic targets were obtained. Moreover, 5 targets with high degree including TNF, ACTB, INS, JUN, and MMP9 were identified as core targets of API for treating AAA. In addition, functional enrichment analysis indicated that API exerted pharmacological effects in AAA by affecting versatile mechanisms, including apoptosis, inflammation, blood fluid dynamics, and immune modulation. Molecular docking results further supported that API had strong affinity with the above core targets. Furthermore, protein level of core targets and related pathways were evaluated in a Cacl2-induced AAA model by using western blot and immunohistochemistry. The experimental validation results demonstrated that API significantly attenuated phosphorylation of JUN and protein level of predicted core targets. Taken together, based on network pharmacological and experimental validation, our study systematically explored associated core targets and potential therapeutic pathways of API for AAA treatment, which could supply valuable insights and theoretical basis for AAA treatment.

Olfactory receptor 2 deficiency protects from experimental abdominal aortic aneurysm formation

Olfactory receptor 2 deficiency protects from experimental abdominal aortic aneurysm formation

The presence of activating IgG Fc receptors in macrophages aggravates the development of experimental abdominal aortic aneurysm

IntroductionAbdominal aortic aneurysm (AAA) is a multifactorial, degenerative disease characterized by progressive aortic dilation and chronic activation of inflammation, proteolytic activity, and oxidative stress in the aortic wall. The immune response triggered by antibodies against antigens present in the vascular wall participates in the formation and progression of AAA through mechanisms not completely understood. This work analyses the function of specific IgG receptors (FcγR), especially those expressed by monocytes/macrophages, in the development of experimental AAA. MethodsIn the elastase-induced AAA model, the abdominal aortas from wildtype and FcγR deficient mice with/without macrophage adoptive transfer were analysed by histology and quantitative PCR. In vitro, RAW 264.7 macrophages were transfected with RNA interference of FcγRIV/CD16.2 or treated with Syk kinase inhibitor before stimulation with IgG immune complexes. ResultsMacrophage adoptive transfer in FcγR deficient mice increased the susceptibility to AAA development. Mice receiving macrophages with functional FcγR exhibited higher aortic diameter increase, higher content of macrophages and B lymphocytes, and upregulated expression of chemokine CCL2, cytokines (TNFα and IL-17), metalloproteinase MMP2, prooxidant enzyme NADPH oxidase-2, and the isoforms FcγRIII/CD16 and FcγRIV/CD16.2. In vitro, both FcγRIV/CD16.2 gene silencing and Syk inhibition reduced cytokines and reactive oxygen species production induced by IC in macrophages. ConclusionsActivation of macrophage FcγR contributes to AAA development by inducing mediators of inflammation, proteolysis, and oxidative stress. Modulation of FcγR or effector molecules may represent a potential target for AAA treatment.

Increased plasma renin by vasodilators promotes the progression of abdominal aortic aneurysm.

Background: It is well-accepted that antihypertensive therapy is the cornerstone of treatment for abdominal aortic aneurysm (AAA) patients with hypertension. Direct-acting vasodilators were used in the treatment of hypertension by directly relaxing vascular smooth muscle but may have destructive effects on the aortic wall by activating the renin-angiotensin system axis. Their roles in AAA disease remain to be elucidated. In this study, we used hydralazine and minoxidil, two classical direct-acting vasodilators, to investigate their influence and potential mechanisms on AAA disease. Methods and results: In this study, we investigated the plasma renin level and plasma renin activity in AAA patients. Simultaneously, age and gender ratio-matched patients diagnosed with peripheral artery disease and varicose veins were selected as the control group using a ratio of 1:1:1. Our regression analysis suggested both the plasma renin level and plasma renin activity are positively associated with AAA development. In view of the well-established relationship between direct-acting vasodilators and increased plasma renin concentration, we established a porcine pancreatic elastase-infused AAA mouse model, followed by oral administration of hydralazine (250mg/L) and minoxidil (120mg/L) to investigate effects of direct-acting vasodilators on AAA disease. Our results suggested both hydralazine and minoxidil promoted the progression of AAA with increased aortic degeneration. Mechanistically, the vasodilators aggravated aortic inflammation by increased leukocyte infiltration and inflammatory cytokine secretion. Conclusion and relevance: The plasma renin level and plasma renin activity are positively associated with AAA development. Direct vasodilators aggravated experimental AAA progression, which raised cautionary concerns about their applications in AAA disease.

Pharmacological Inhibition of Gasdermin D Suppresses Angiotensin II-Induced Experimental Abdominal Aortic Aneurysms.

Gasdermin D, a molecule downstream of the nucleotide-binding oligomerization domain-like receptor family pyrin domain containing inflammasome, forms the membrane pore for the secretion of interleukin (IL)-1β and IL-18, and also mediates pyroptosis. This study was to explore the influence of treatment with disulfiram, a small molecule inhibitor to gasdermin D, on the formation and progression of experimental abdominal aortic aneurysms (AAA). AAAs were induced in 10-week-old male apolipoprotein E deficient mice by subcutaneous infusion of angiotensin II (1000 ng/min/kg body weight) for 28 days via osmotic minipumps. Three days prior to angiotensin II infusion, disulfiram (50 mg/kg) or an equal volume of saline as the vehicle control was administered daily via oral gavage. The influence on experimental AAAs was analyzed by serial measurements of aortic diameters via ultrasonography, grading AAA severity and histopathology at sacrifice. Serum IL-1β and IL-18 levels, systolic blood pressure, total cholesterol, and triglyceride were also measured. Additional experiments assayed the influences on the cell viability and IL-1β secretion of in vitro activated macrophages. Disulfiram significantly reduced the enlargement, incidence, and severity of angiotensin II-induced experimental AAAs with attenuation of medial elastin breaks, mural macrophage accumulation, and systolic blood pressure. The AAA suppression was also associated with reduced systemic levels of IL-1β but not IL-18. However, disulfiram treatment had no impact on body weight gain and lipid levels in aneurysmal mice. Additionally, disulfiram treatment also markedly reduced the secretion of IL-1β from activated macrophages with a limited effect on cell viability in vitro. Gasdermin D inhibition by disulfiram attenuated angiotensin II-induced experimental AAAs with reduced systemic IL-1β levels and in vitro activated macrophage IL-1β secretion. Our study suggests that pharmacological gasdermin D inhibition may have translational potential for limiting clinical AAA progression.

IL-1 in Abdominal Aortic Aneurysms.

Abdominal Aortic Aneurysms (AAA) remain a clinically devastating disease with no effective medical treatment therapy. AAAs are characterized by immune cell infiltration, smooth muscle cell apoptosis, and extracellular matrix degradation. Interleukin-1 (IL-1) has been shown to play role in AAA associated inflammation through immune cell recruitment and activation, endothelial dysfunction, production of reactive oxygen species (ROS), and regulation of transcription factors of additional inflammatory mediators. In this review, we will discuss the principles of IL-1 signaling, its role in AAA specific inflammation, and regulators of IL-1 signaling. Additionally, we will discuss the influence of genetic and pharmacological inhibitors of IL-1 on experimental AAAs. Evidence suggests that IL-1 may prove to be a potential therapeutic target in the management of AAA disease.

Targeting mitochondrial stress with SS31 prevents experimental abdominal aortic aneurysm: crosstalk with ER stress.

Background and purpose mitochondrial dysfunction and inflammation contribute to the pathophysiology of a myriad of cardiovascular diseases. Additionally, the deleterious crosstalk of mitochondria and persistent endoplasmic reticulum (ER) stress triggers oxidative stress, which is deeply involved in the development of vascular diseases. The purpose of this work was to determine whether inhibition of mitochondrial stress reduces aneurysm development in angiotensin II (Ang II)-infused apolipoprotein-E-deficient (ApoE-/- ) mice and its effect on ER stress. Experimental approach and results the mitochondria-targeted tetrapeptide SS31 ameliorated mitochondrial dysfunction and the enhanced expression of ER stress markers triggered by Ang II in ApoE-/- mice and limited plasmatic and vascular ROS levels. Interestingly, SS31 improved survival, reduced the incidence and severity of abdominal aortic aneurysm (AAA) and the AngII-induced increase of aortic diameter evaluated by ultrasonography, resembling the response triggered by the classic ER stress inhibitors TUDCA and PBA. The disorganization of extracellular matrix, the increased expression of metalloproteinases and pro-inflammatory markers and the infiltration of immune cells induced by AngII in the abdominal aorta were effectively reduced by both, SS31 and ER inhibitors. Further, CHOP deficiency in ApoE-/- mice attenuated the AngII-mediated increase in vascular diameter and the incidence of AAA, suggesting its contribution to the favorable response induced by ER stress inhibition. CONCLUSIONS: our data demonstrate that inhibition of mitochondrial stress by SS31 limits AAA formation and increases survival through a reduction of vascular remodeling, inflammation and ROS, and support that the attenuation of ER stress also contribute to the favorable response elicited by SS31.

Purinergic receptor P2X7 contributes to abdominal aortic aneurysm development via modulating macrophage pyroptosis and inflammation

The purinergic receptor P2X7 has been established as an important mediator of inflammation and participates in a variety of cardiovascular diseases including atherosclerosis, however, its role in abdominal aortic aneurysms (AAA) remains unclear. In this study, we demonstrate that P2X7 plays essential roles in AAA development via modulating macrophage pyroptosis and inflammation. P2X7 is highly expressed in human AAA specimen, as well as in experimental murine AAA lesions (both CaCl2- and Angiotensin II-induced AAA models), and it mainly confines in macrophages. Furthermore, P2X7 deficiency or pharmacological inhibition with its antagonist could significantly attenuate aneurysm formation in experimental murine AAA models, while P2X7 agonist could promote AAA development. The caspase-1 activity, matrix metalloproteinase (MMP) activity, reactive oxygen species (ROS) production and pro-inflammatory gene expression were significantly reduced in experimental AAA lesions in mice with P2X7 deficiency or inhibition. Mechanistically, macrophage P2X7 can mediate the activation of NLRP3 inflammasome and activate its downstream caspase-1 to initiate the pyroptosis pathway. After caspase-1 activation, it further cleaves pro-interleukin (IL)-1β and gasdermin D (GSDMD). Consequently, the N-terminal fragment of GSDMD forms pores on the cell membrane, leading to macrophage pyroptosis and release of the pro-inflammatory factor IL-1β. The resulting vascular inflammation further leads to the upregulation of MMP and ROS, thereby promoting AAA development. In summary, these data identify P2X7-mediated macrophage pyroptosis signaling pathway as a novel contributory mechanism of AAA formation.

Genetic deficiency of protein inhibitor of activated STAT3 suppresses experimental abdominal aortic aneurysms.

Signal transducer and activator of transcription (STAT) signaling is critical for the pathogenesis of abdominal aortic aneurysms (AAAs). Though protein inhibitor of activated STAT3 (PIAS3) negatively modulates STAT3 activity, but its role in AAA disease remains undefined. AAAs were induced in PIAS3 deficient (PIAS3-/-) and wild type (PIAS3+/+) male mice via transient intra-aortic elastase infusion. AAAs were assessed by in situ measurements of infrarenal aortic external diameters prior to (day 0) and 14 days after elastase infusion. Characteristic aneurysmal pathologies were evaluated by histopathology. Fourteen days following elastase infusion, aneurysmal aortic diameter was reduced by an approximately 50% in PIAS3-/- as compared to PIAS3+/+ mice. On histological analyses, PIAS3-/- mice showed less medial elastin degradation (media score: 2.5) and smooth muscle cell loss (media score: 3.0) than those in PIAS3+/+ mice (media score: 4 for both elastin and SMC destruction). Aortic wall leukocyte accumulation including macrophages, CD4+ T cells, CD8+ T cells and B cells as well as mural neovessel formation were significantly reduced in PIAS3-/- as compared to PIAS3+/+ mice. Additionally, PIAS3 deficiency also downregulated the expression levels of matrix metalloproteinases 2 and 9 by 61% and 70%, respectively, in aneurysmal lesion. PIAS3 deficiency ameliorated experimental AAAs in conjunction with reduced medial elastin degradation and smooth muscle cell depletion, mural leukocyte accumulation and angiogenesis.

Platelet pannexin-1 channels modulate neutrophil activation and migration but not the progression of abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) is a common disease and highly lethal if untreated. The progressive dilatation of the abdominal aorta is accompanied by degradation and remodeling of the vessel wall due to chronic inflammation. Pannexins represent anion-selective channels and play a crucial role in non-vesicular ATP release to amplify paracrine signaling in cells. Thus, pannexins are involved in many (patho-) physiological processes. Recently, Panx1 channels were identified to be significantly involved in abdominal aortic aneurysm formation through endothelial derived Panx1 regulated inflammation and aortic remodeling. In platelets, Panx1 becomes activated following activation of glycoprotein (GP) VI. Since platelets play a role in cardiovascular diseases including abdominal aortic aneurysm, we analyzed the contribution of platelet Panx1 in the progression of abdominal aortic aneurysm. We detected enhanced Panx1 plasma levels in abdominal aortic aneurysm patients. In experimental abdominal aortic aneurysm using the pancreatic porcine elastase (PPE) mouse model, a major contribution of platelet Panx1 channels in platelet activation, pro-coagulant activity of platelets and platelet-mediated inflammation has been detected. In detail, platelets are important for the migration of neutrophils into the aortic wall induced by direct cell interaction and by activation of endothelial cells. Decreased platelet activation and inflammation did not affect ECM remodeling or wall thickness in platelet-specific Panx1 knock-out mice following PPE surgery. Thus, aortic diameter expansion at different time points after elastase infusion of the aortic wall was unaltered in platelet-specific Panx1 deficient mice suggesting that the modulation of inflammation alone does not affect abdominal aortic aneurysm formation and progression. In conclusion, our data strongly supports the role of platelets in inflammatory responses in abdominal aortic aneurysm via Panx1 channels and adds important knowledge about the significance of platelets in abdominal aortic aneurysm pathology important for the establishment of an anti-platelet therapy for abdominal aortic aneurysm patients.

The gut microbiota in experimental abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) is a life-threatening disease and there are no effective treatments to inhibit aneurysm progression and rupture. The gut microbiota has been increasingly recognized, as a new therapeutic target, because of its role in host homeostasis. However, the role of the gut microbiota in AAA has not been clarified. Therefore, we performed 16S rRNA analysis to determine and compare the composition of the gut microbiota between AAA and control groups. We used the classical angiotensin-II induced AAA mouse model to investigate the role of gut microbiota and abdominal aortic aneurysm. The mice were randomly assigned to 2 groups: the control (n = 7) group received saline (vehicle), while the AAA (n = 13) group received solutions of Ang II. Aortic tissue and fecal samples were harvested 28 days after infusion. Fecal samples were analyzed by 16S rRNA sequencing. The levels of Oscillospira, Coprococcus, Faecalibacterium prausnitzii, Alistipes massiliensis, and Ruminococcus gnavus were increased in the AAA group, while those of Akkermansia muciniphila, Allobaculum, and Barnesiella intestinihominis were increased in the control group. Furthermore, network analysis and ZiPi score assessment highlighted species in the phylum Bacteroidetes as the keystone species. PICRUSt2 analysis revealed that PWY-6629 (a super pathway of L-tryptophan biosynthesis), PWY-7446 (sulfoglycolysis), and PWY-6165 [chorismate biosynthesis II (archaea)] may-be involved in the metabolic pathways that contribute to AAA formation, and E. coli/Shigella may be the key bacteria that influence those three pathways. Alterations in the gut microbiota may be associated with the formation of AAA. Akkermansia and Lactobacillus were significantly decreased in the AAA group, but the keystone species in the phylum Bacteroidetes and the metabolic products of these bacteria should be given more attention in AAA formation research.

Pharmacologic inhibition by spironolactone attenuates experimental abdominal aortic aneurysms.

Abdominal aortic aneurysms (AAA) are characterized by vascular inflammation and remodeling that can lead to aortic rupture resulting in significant mortality. Pannexin-1 channels on endothelial cells (ECs) can modulate ATP secretion to regulate the pathogenesis of AAA formation. Our hypothesis focused on potential of spironolactone to inhibit EC-mediated ATP release for the mitigation of AAA formation. A topical elastase AAA model was used initially in C57BL/6 (wild-type; WT) male mice. Mice were administered either a vehicle control (saline) or spironolactone and analyzed on day 14. In a second chronic AAA model, mice were subjected to elastase and β-aminopropionitrile (BAPN) treatment with/without administration of spironolactone to pre-formed aneurysms starting on day 14 and analyzed on day 28. Aortic diameter was evaluated by video micrometry and aortic tissue was analyzed for cytokine expression and histology. ATP measurement and matrix metalloproteinase (MMP2) activity was evaluated in aortic tissue on days 14 or -28. In vitro studies were performed to evaluate the crosstalk between aortic ECs with macrophages or smooth muscle cells. In the elastase AAA model, spironolactone treatment displayed a significant decrease in aortic diameter compared to elastase-treated controls on day 14. A significant increase in smooth muscle α-actin expression as well as decrease in elastic fiber disruption and immune cell (macrophages and neutrophils) infiltration was observed in mice treated with spironolactone compared to saline-treated controls. Spironolactone treatment also significantly mitigated pro-inflammatory cytokine expression, MMP2 activity and ATP content in aortic tissue compared to controls. Moreover, in the chronic AAA model, spironolactone treatment of pre-formed aneurysms significantly attenuated vascular inflammation and remodeling to attenuate the progression of AAAs compared to controls. Mechanistically, in vitro data demonstrated that spironolactone treatment attenuates extracellular ATP release from endothelial cells to mitigate macrophage activation (IL-1β and HMGB1 expression) and smooth muscle cell-dependent vascular remodeling (MMP2 activity). These results demonstrate that spironolactone can mitigate aortic inflammation and remodeling to attenuate AAA formation as well as decrease growth of pre-formed aneurysms via inhibition of EC-dependent ATP release. Therefore, this study implicates a therapeutic application of spironolactone in the treatment of AAAs.