Abdominal Aortic Aneurysm Tissue Research Articles

Hyperglycemia inhibits AAA expansion: examining the role of lysyl oxidase.

Abdominal aortic aneurysm (AAA) is a common, progressive, and potentially fatal dilation of the most distal aortic segment. Multiple studies with longitudinal follow-up of AAA have identified markedly slower progression among patients affected with diabetes. Understanding the molecular pathway responsible for the growth inhibition could have implications for therapy in nondiabetic patients with AAA. Toward this end, we investigated the effects of hyperglycemia in a murine model of AAA and a carefully monitored cohort of patients with AAA from the Noninvasive Treatment of AAA-Clinical Trial (NTA3CT). In mice with hyperglycemia, AAA growth was inhibited to a similar degree (∼30%) as seen in patients with diabetes. AAA growth correlated inversely to levels of hyperglycemia in mice and patients with AAA. Inhibiting lysyl oxidase (LOX) activity increases aneurysm growth and matrix degradation in this model. Hyperglycemia increased LOX concentration in aortic smooth muscle cells (SMCs) but not in murine AAA tissue. Inhibiting LOX activity completely blocked the growth-inhibitory effect of hyperglycemia. Lysyl oxidase-like 2 (LOXL2), the primary arterial isoform of LOX, is expressed in the same area as type IV collagen along the outer media in murine AAA tissue. There is a significant inverse correlation between LOXL2 and AAA growth rates in patients. Taken together, these studies suggest a role for LOXL2-mediated type IV collagen crosslinking in slowing AAA growth in the setting of hyperglycemia.NEW & NOTEWORTHY AAA grows slower in patients affected by diabetes. This growth inhibition is lost when the enzyme lysyl oxidase (LOX) is blocked in diabetic mice. The predominant arterial isoform of LOX, LOX-like 2 (LOXL2), overlaps with type IV collagen in the outer media of murine aneurysm tissue. Circulating LOXL2 correlates inversely with AAA growth in patients. Type IV collagen cross-linking by LOXL2 may play a role in the AAA growth inhibition associated with diabetes.

Inhibition of vascular smooth muscle cell PERK/ATF4 ER stress signaling protects against abdominal aortic aneurysms.

Abdominal aortic aneurysms (AAA) are a life-threatening cardiovascular disease for which there is a lack of effective therapy preventing aortic rupture. During AAA formation, pathological vascular remodeling is driven by vascular smooth muscle cell (VSMC) dysfunction and apoptosis, for which the mechanisms regulating loss of VSMCs within the aortic wall remain poorly defined. Using single-cell RNA-Seq of human AAA tissues, we identified increased activation of the endoplasmic reticulum stress response pathway, PERK/eIF2α/ATF4, in aortic VSMCs resulting in upregulation of an apoptotic cellular response. Mechanistically, we reported that aberrant TNF-α activity within the aortic wall induces VSMC ATF4 activation through the PERK endoplasmic reticulum stress response, resulting in progressive apoptosis. In vivo targeted inhibition of the PERK pathway, with VSMC-specific genetic depletion (Eif2ak3fl/fl Myh11-CreERT2) or pharmacological inhibition in the elastase and angiotensin II-induced AAA model preserved VSMC function, decreased elastin fragmentation, attenuated VSMC apoptosis, and markedly reduced AAA expansion. Together, our findings suggest that cell-specific pharmacologic therapy targeting the PERK/eIF2α/ATF4 pathway in VSMCs may be an effective intervention to prevent AAA expansion.

TRAF6 promotes abdominal aortic aneurysm development by activating macrophage pyroptosis via the NLRP3/Caspase1/GSDMD pathway.

Abdominal aortic aneurysm represents a critical pathology of the aorta that currently lacks effective pharmacological interventions. TNF receptor-associated factor 6 (TRAF6) has been established to be involved in cardiovascular diseases such as atherosclerosis, hypertension, and heart failure. However, its role in abdominal aortic aneurysm (AAA) remains unclear. This study aimed to explore the role of TRAF6 on AAA formation and its underlying mechanisms. Single-cell RNA sequencing of human AAA tissues demonstrated that TRAF6 was significantly upregulated in aortic macrophages. Moreover, overexpression of TRAF6 promotes AAA formation in elastase-induced C57BL/6 mice, while TRAF6 pharmacological inhibition could attenuate AAA development. Consistently, inhibition of TRAF6 in macrophages through invitro methods notably limits their pyroptosis, while also diminishing proinflammatory responses in these cells. Mechanistically, TRAF6 can modulate macrophage pyroptosis through the NLRP3/Caspase1/GSDMD signaling pathway. Our study highlights the crucial role of the TRAF6/NLRP3/Caspase1/GSDMD axis in macrophage pyroptosis and AAA, offering potential biomarkers and therapeutic targets for AAA.

Inhibition of Abdominal Aortic Aneurysm Progression Through the CXCL12/CXCR4 Axis via MiR206-3p Sponge.

Notably, the C-X-C Motif Chemokine Ligand 12/C-X-C Chemokine Receptor Type 4 (CXCL12/CXCR4) signalling pathway's activation is markedly increased in a mouse model of abdominal aortic aneurysms (AAA). Nonetheless, the precise contribution of this pathway to AAA development remains to be elucidated. The AAA mouse model was induced by local incubation with elastase and oral administration of β-aminopropionitrile. The activity level of the CXCL12/CXCR4 axis was evaluated in both human AAA patients and the mouse model. Smooth muscle cell lineage tracing determined the expression and localisation of CXCR4 in normal aorta and AAA tissue. By transfecting the MiR206-3p sponge to reduce the level of MiR206-3p in AAA, the effects of the CXCL12/CXCR4 pathway on AAA progression as well as the apoptosis and phenotypic transformation of vascular smooth muscle cells (VSMCs) were studied invivo and invitro. Single-cell RNA sequencing analysis, serum ELISA, and invivo experiments indicate a pronounced activation of the CXCL12/CXCR4 axis in both AAA patients and the mouse model. Specific blocking of the CXCL12/CXCR4 axis significantly inhibited further expansion and rupture of the abdominal aorta and reduced the infiltration of inflammatory cells in the aorta and inhibited the phenotypic transformation of contractile VSMCs into a macrophage-like state. Our findings propose that MiR206-3p sponge represents an innovative therapeutic strategy to attenuate AAA progression and rupture risk, primarily through the suppression of the CXCL12/CXCR4 signalling pathway.

Inhibition of GPR4 Attenuates the Formation of Abdominal Aortic Aneurysm Through Inhibiting the SP-1/VEGF-A Signaling.

Abdominal aortic aneurysm (AAA) is a severe cardiovascular disease (CVD) that is partly attributable to endothelial dysfunction, inflammatory response, and angiogenesis. G protein-coupled receptor 4 (GPR4), a proton-sensitive G protein-coupled receptor that is abundantly expressed in vascular endothelial cells, has been associated with numerous physiological functions. Nevertheless, its potential involvement in the development of AAA remains unexplored. In this study, we examined the impact of GPR4 deletion on the development of AAA in ApoE-deficient mice. The mice were categorized into four distinct groups: the ApoE-/- with saline group, the ApoE-/-GPR4-/- with saline group, the ApoE-/- with Ang II group, and the ApoE-/-GPR4-/- with Ang II group. AAA were induced in the ApoE-/- mice through the perfusion of angiotensin II (Ang II). Notably, GPR4 was substantially elevated in the AAA tissues from both human subjects and experimental mice. The deletion of GPR4 substantially decreased the formation of Ang II-induced AAA, damages to elastin, and the expression of aortic inflammatory cytokines interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α), as well as vascular endothelial growth factor A/vascular endothelial growth factor receptor 2 (VEGF-A/VEGF-R2), in ApoE-/- mice. Human aortic endothelial cells (HAECs) were transfected with lenti-viral GPR4 shRNA and subsequently stimulated with Ang II. Our findings indicate that the knockout of GPR4 attenuated Ang II-induced angiogenic tube formation in HAECs by decreasing the expression of VEGF-A and VEGF-R2. Furthermore, GPR4 knockout also hindered the activation of specificity protein-1 (SP-1) by reducing its expression and transcriptional activity. Notably, the overexpression of SP-1 reversed the inhibitory effects of GPR4 knockout on angiogenic tube formation and the expression of VEGF-A/VEGF-R2. This suggests that the protective effects of GPR4 knockout are achieved through the inhibition of SP-1. In summary, the absence of GPR4 impeded AAA formation, indicating that GPR4 could potentially serve as a therapeutic target for AAA.

Prenyl diphosphate synthase subunit 2 is downregulated in abdominal aortic aneurysm and retards the progression of abdominal aortic aneurysm

Objective: Abdominal aortic aneurysm (AAA) is a complex and fatal vascular disease for which specific treatments are still lacking. This study explored the effect and possible mechanisms of prenyl diphosphate synthase subunit 2 (PDSS2) on angiotensin II (Ang II)-induced AAA in human vascular smooth muscle cells (VSMCs). Material and Methods: The AAA cell model was established by treating VSMCs with 1 μM Ang II for 24 h. The effect of Ang II on VSMC viability was detected by cell counting kit-8 assay. The role of PDSS2 on VSMC proliferation was examined using the 5-ethynyl-2'-deoxyuridine method. The influence of Ang II and PDSS2 on VSMC apoptosis was analyzed by flow cytometry. The expression changes of PDSS2, apoptosis-related proteins, and phosphatidylinositol 3 kinase/protein kinase B/mechanistic target of rapamycin (PI3K/AKT/mTOR) pathway-related proteins were detected by Western blot analysis. Results: After treatment with Ang II, the VSMCs showed decreased viability and increased apoptosis (P < 0.01). PDSS2 expression was low in the AAA tissues and Ang II-treated VSMCs (P < 0.01). PDSS2 promoted the proliferation and blocked the apoptosis of Ang II-treated VSMCs, and si-PDSS2 showed the opposite effect (P < 0.01). PDSS2 also decreased the levels of p-mTOR, p-AKT, and p-PI3K, which, in turn, were increased by si-PDSS2 (P < 0.01). Conclusion: PDSS2 was downregulated in AAA and retarded the progression of VSMCs partially through the PI3K/AKT/mTOR pathway. This work explored the molecular mechanism of PDSS2 in the prevention, diagnosis, and treatment of AAA.

CircFNDC3B inhibits vascular smooth muscle cells proliferation in abdominal aortic aneurysms by targeting the miR-1270/PDCD10 axis

Objectives. This study investigated the role and underlying regulatory mechanisms of circular RNA fibronectin type III domain containing 3B (circFNDC3B) in abdominal aortic aneurysm (AAA). Methods. The expression of circFNDC3B in AAA and normal tissues was assessed by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). To evaluate the biological functions of circFNDC3B, assays were employed including 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), flow cytometry, and Caspase-3 activity assays. Additionally, RNA immunoprecipitation (RIP), dual-luciferase reporter assay, Western blotting, and rescue experiments were utilized to elucidate the molecular mechanism of circFNDC3B. Results. Our findings revealed a significant upregulation of circFNDC3B expression in AAA clinical specimens compared to normal tissues. Functionally, overexpression of circFNDC3B inhibited vascular smooth muscle cells (VSMCs) proliferation and induced apoptosis, contributing to AAA formation in the Ang II-induced AAA model. Mechanistically, circFNDC3B acted as a molecular sponge for miR-1270, leading to the upregulation of programmed cell death 10 (PDCD10). Decreased expression of PDCD10 abrogated the -promoting effects of circFNDC3B overexpression on AAA development. Conclusions. This study demonstrates that circFNDC3B promotes the progression of AAA by targeting the miR-1270/PDCD10 pathway. Our findings suggest that circFNDC3B as well as miR-1270/PDCD10 pathway may serve as a potential therapeutic target for AAA treatment.

Revealing the roles of IL-7R in abdominal aortic aneurysm through integrated analysis of single-cell RNA-seq and bulk RNA-seq.

Abdominal aortic aneurysm (AAA) is a common cardiovascular disease in the elderly, but there are still no therapeutic targets for this disease. In this study, we collected and analyzed bulk RNA sequencing (bulk RNA-seq) and single-cell RNA sequencing (scRNA-seq) data of AAA from the Gene Expression Omnibus (GEO) database. The immune infiltration-related genes were identified and categorized into various cell types, revealing potential key genes and pathways. Examination of three bulk RNA-seq datasets revealed a total of 4087 differentially expressed genes. The expression levels of the immune-related genes IL-7R were significantly elevated in AAA tissues across all three datasets. Furthermore, scRNA-Seq analysis revealed increased expression of IL-7R in CD4+ memory cells within AAA tissues. Immunofluorescence staining corroborated these findings, demonstrating increased expression of IL-7R in CD4+ T cells in AAA tissues. In vitro, activation of IL-7R elevated the activation of JAK/STAT pathway and phenotypic switching in SMCs, while inhibition of IL-7R abolished these effects and suppressed the secretion of IFN-γ. In conclusion, the activation of IL-7R in CD4+ T cells is a key contributor to the pathogenesis of AAA, as it can promote secretion of IFN-γ via JAK/STAT pathway and induce phenotypic switching of SMCs.

Disruption of PCSK9 Suppresses Inflammation and Attenuates Abdominal Aortic Aneurysm Formation.

Abdominal aortic aneurysm (AAA) is a chronic vascular inflammatory disease without effective medications. PCSK9 (proprotein convertase subtilisin/kexin 9), a serine protease from the proprotein convertase family, has recently been associated with AAA in human genome-wide association studies. However, its role in AAA is unknown. Transcriptional and histological expression of PCSK9 was examined in AAA tissues and healthy controls. The impact of PCSK9 deletion and inhibition on AAA formation was assessed in mice with hyperlipidemia and Ang II (angiotensin II) overproduction. AAA lesion morphology was assessed by tissue staining. MMP (matrix metalloproteinase) activity was evaluated by gelatin zymography, and leukocyte-vessel wall interaction was monitored by intravital microscopy. RNA sequencing was used to characterize the downstream signaling of PCSK9. PCSK9 expression was upregulated and colocalized with macrophages in human and mouse AAAs. Pcsk9 deletion attenuated AAA formation, improved survival, and decreased systemic inflammation, without altering circulating cholesterol levels. Pcsk9 deficiency reduced aortic infiltration of macrophages and elastin degradation, without affecting vascular smooth muscle cell apoptosis and proliferation. Mechanistically, PCSK9 was essential in leukocyte-endothelium adhesion and expression of proinflammatory cytokines and MMP9 by macrophages. RNA sequencing of stimulated macrophages revealed that Pcsk9 deficiency upregulated histone deacetylase SIRT1 (sirtuin-1) and suppressed NF-κB (nuclear factor-κB) inflammatory signaling. SIRT1 inhibition attenuated the proinflammatory actions of PCSK9. Furthermore, administration of PCSK9 small interfering RNA or antibody constrained AAA formation/progression and inhibited vascular inflammation. PCSK9 critically mediates macrophage inflammation and elastin degradation, promoting AAA formation. PCSK9 inhibitors bear a promise to curtail AAA, beyond being used as cholesterol-lowering drugs.

Abstract 4120741: Estrogen Regulates Abdominal Aortic Aneurysm Tregs via CD45 + Fibroblast-derived IL-33

Background: Sexual dimorphisms manifest during the progression of abdominal aortic aneurysm (AAA). Regulatory T cells (Tregs) are a subset of T cells characterized by their immunosuppressive and tissue-repairing functions, therefore playing a protective role in AAA. Aims: We aim to elucidate the mechanism underlying sexual dimorphism in Tregs presented in AAA aortas, which is significant for unraveling the pathogenesis of AAA and developing gender-specific therapeutic approaches. Methods and Results: AAA mice were induced by receiving porcine pancreatic elastase. By flow cytometry, we observed that ovariectomy (OVX) increased the ratio of Tregs in female mice, while estrogen led to a decrease in aortic Tregs. Additionally, compared to littermate controls, estrogen receptor α (ERα) KO mice exhibited an increase in Treg ratio. Similar results were observed for the ST2 + Treg ratio. IL-33, as an alarming molecule, can promote the maintenance of ST2 + Tregs in tissues. Western blot revealed lower IL-33 expression levels in female mice than in male mice. By analyzing the IL-33 expression profiles of various cells in AAA tissue, we found that IL-33 mainly originates from fibroblasts. We found two subpopulations of fibroblasts, CD45 + and CD45 - fibroblasts. Immunofluorescence on the aortas verified the presence of cells co-expressing CD45 and PDGFRα in AAA fragments. Furthermore, IL-33 expression only showed gender differences in CD45 + fibroblasts. Adoptive transfer of CD45.1 + mouse bone marrow macrophages into CD45.2 + mice revealed that aortic CD45.1 + cells could regain the PDGFRα phenotype, with a lower proportion of female PDGFRα + cells in CD45.2 - CD45.1 + cells than in male mice. Lyz2(cre/+); Rosa26(tdtomato/+) mice showed the presence of lyz2-tdtomato + cells expressing PDGFRα in the aortic adventitia after AAA induction. This suggests that CD45 + fibroblasts are partially derived from bone marrow macrophages, which can migrate to aneurysms and acquire a PDGFRα phenotype. Conclusions: Female sex hormones negatively regulate CD45 + fibroblast formation and IL-33 secretion between female and male mice during AAA. This resulted in reduced Treg function in female mice compared to male mice.

Abstract 4137693: CC-Chemokine Receptor 2 Inhibition Prevents Monocyte/Macrophages Recruitment in Abdominal Aortic Aneurysms

Intro: Abdominal aortic aneurysm (AAA) is a prevalent condition in the elderly, with a significant risk of mortality if it ruptures. Despite this, there is currently no effective medical therapy to prevent AAA growth and rupture. The monocyte chemoattractant protein (MCP-1) / C-C chemokine receptor type 2 (CCR2) axis appears to play a role in AAA development. We demonstrated the potential of CCR2 as a theranostic marker for predicting AAA rupture, along with the effectiveness of a CCR2 inhibitor (RS-504393) in mitigating rupture. We further hypothesized that CCR2 inhibition can impact monocytes/macrophages trafficking in aortic tissue. Mehtods: Sprague-Dawley rats (N=54) underwent intraluminal aortic exposure to porcine pancreatic elastase (PPE) to induce aneurysmal degeneration. To stimulate AAA rupture, rats also recieved β-aminopropionitrile (BAPN). After 3 days of PPE exposure, a group of rats with AAA were administered a CCR2 inhibitor (CCR2i) either daily until the end of the study (CCR2i Full), or discontinued at day 5 (CCR2i Short), while the remaining rats proceeded without intervention (Fig1A). Ultrasound (12MHz) was performed at 7&14 days to assess diameter, while another cohort of animals were euthanized at 6 days, prior to rupture, for single-cell RNA sequencing analysis. The relative content of inflammatory cells within the aortic tissue was analyzed. Results: Both CCR2i Short and CCR2i Full treated, exhibited a significant reduction in AAA diameter at day 7 and 14 compared to controls (p<0.05; Fig1B&C). Notably, rats treated with CCR2i Full did not experience any AAA ruptures, while controls had a 56% risk of rupture. In contrast, CCR2i Short delayed the occurrence of ruptures without reducing the rupture rate (p<0.01; Fig1D). Single-cell RNA seq analysis of aortic tissue revealed that CCR2 inhibition led to a decrease in the number of recruited monocytes/macrophages. Cell recruitment was also relative to the duration of CCR2i treatment (Fig1E-G). Conclusions: Our study findings highlight the impact of CCR2-targeting in reducing AAA growth and the risk of rupture. Interestingly, length of CCR2i was a crucial variable for AAA rupture prevention. Furthermore, treatment length impacted the relative recruitment of monocytes/macrophages to AAA tissue, highlighting the role these cells in AAA disease pathology. Future analysis of CCR2+ inflammatory cell subgroups may help elucidate how CCR2 mechanistically modulates AAA progression and rupture risk.

EUGENOL RESTRAINS ANGIOTENSIN II-INDUCED DEATH, INFLAMMATION AND FERROPTOSIS OF VASCULAR SMOOTH MUSCLE CELLS BY TARGETING STAT3/HMGB2 AXIS.

Background: Eugenol has been found to inhibit a variety of disease processes, including abdominal aortic aneurysm (AAA) formation. However, the specific role and the underlying molecular mechanism of Eugenol in AAA progression need to be further revealed. Methods: Vascular smooth muscle cells (VSMCs) were pretreated with Eugenol, followed by treated with Angiotensin II (Ang-II). VSMCs were transfected with HMGB2 siRNA or overexpression vector and treated with Ang-II to confirm the effect of HMGB2 on AAA progression. Cell proliferation and death were determined using cell counting kit 8 assay, 5-ethynyl-2'-deoxyuridine assay, and flow cytometry. Inflammatory factors were examined by ELISA. Fe 2+ , glutathione, and malondialdehyde levels were tested to evaluate cell ferroptosis. The protein levels of ferroptosis-related markers, high mobility group box 2 (HMGB2), and STAT3 were measured using western blot. Human AAA tissues and normal abdominal aortic tissues were collected to detect HMGB2 mRNA expression by quantitative real-time PCR. The interaction between HMGB2 and STAT3 was confirmed by chromatin immunoprecipitation assay and dual-luciferase reporter assay. Results: Eugenol enhanced VSMCs proliferation, while restrained Ang-II-induced death, inflammation, and ferroptosis. HMGB2 was upregulated in AAA tissues and Ang-II-induced VSMCs, and Eugenol significantly decreased HMGB2 expression. HMGB2 knockdown reduced Ang-II-induced VSMCs death, inflammation, and ferroptosis, Besides, HMGB2 overexpression abolished the effect of Eugenol on Ang-II-induced VSMCs injury. Transcription factor STAT3 bound to HMGB2 promoter region to increase its expression. In addition, Eugenol decreased STAT3 expression to regulate HMGB2. Conclusion: Eugenol could slow down the development of AAA, which might be achieved by regulating STAT3/HMGB2 axis.

Activation of nuclear receptor pregnane-X-receptor protects against abdominal aortic aneurysm by inhibiting oxidative stress

Abdominal aortic aneurysm (AAA) is a life-threatening condition, but effective medications to prevent its progression and rupture are currently lacking. The nuclear receptor pregnane-X-receptor (PXR) plays a crucial role in vascular homeostasis. However, the role of PXR in AAA development remains unknown. We first detected the PXR expression in human and murine AAA tissues by RT-qPCR and Western blot. To investigate the potential role of PXR in the development of AAA, we used adeno-associated virus-mediated overexpression of PXR and pharmacological activation of PXR by ginkgolide A (GA) in mouse AAA models induced by both angiotensin II (AngII) and calcium phosphate [Ca3(PO4)2]. The underlying mechanism was further explored using RNA-sequencing and molecular biological analyses. We found a significant decrease in both mRNA and protein levels of PXR in both human and murine aortic smooth muscle cells from AAA tissues, accompanied with phenotypic switching of vascular smooth muscle cell and increased oxidative stress. PXR overexpression in abdominal aortas and GA treatment successfully suppressed AAA formation in both mouse AAA models. RNA-sequencing data revealed that PXR activation inhibited gamma-aminobutyric acid type A receptor subunit alpha3 (GABRA3) expression. Additional mechanistic studies identified that PXR suppressed AAA through mitigating GABRA3-induced reactive oxygen species (ROS) generation and subsequent phosphorylation of c-Jun N-terminal kinase (JNK). Interestingly, p-JNK was found to induce ubiquitin-proteasome degradation of PXR. In summary, our data unveiled, for the first time, the protective role of PXR against AAA pathogenesis by inhibiting oxidative stress. These findings suggested PXR as a promising therapeutic target for AAA.

Single-Cell RNA Sequencing Deconstructs the Distribution of Immune Cells Within Abdominal Aortic Aneurysms in Mice.

Inflammation is a key component in the development of abdominal aortic aneurysm (AAA), yet insights into the roles of immune cells and their interactions in this process are limited. Using single-cell RNA transcriptomic analysis, we deconstructed the CD45+ cell population in elastase-induced murine AAA at the single-cell level. We isolated each group of immune cells from murine AAA tissue at different time points and divided them into several subtypes, listed the remarkable differentially expressed genes, explored the developmental trajectories of immune cells, and demonstrated the interactions among them. Our findings reveal significant differences in several immune cell subsets, including macrophages, dendritic cells, and T cells, within the AAA microenvironment compared with the normal aorta. Especially, conventional dendritic cell type 1 exclusively existed in the AAA tissue rather than the normal aortas. Via CellChat analysis, we identified several intercellular communication pathways like visfatin, which targets monocyte differentiation and neutrophil extracellular trap-mediated interaction between neutrophils and dendritic cells, which might contribute to AAA development. Some of these pathways were validated in human AAA. Despite the absence of external pathogenic stimuli, AAA tissues develop a complex inflammatory microenvironment involving numerous immune cells. In-depth studies of the inflammatory network shall provide new strategies for patients with AAA.

Exploration of small molecule compounds targeting abdominal aortic aneurysm based on CMap database and molecular dynamics simulation.

The mitigation of abdominal aortic aneurysm (AAA) growth through pharmaceutical intervention offers the potential to avert the perils associated with AAA rupture and the subsequent need for surgical intervention. Nevertheless, the existing effective drugs for AAA treatment are limited, necessitating a pressing exploration for novel therapeutic medications. AAA-related transcriptome data were downloaded from GEO, and differentially expressed genes (DEGs) in AAA tissue were screened for GO and KEGG enrichment analyses. Small molecule compounds and their target proteins with negative connectivity to the AAA expression profile were predicted in the Connectivity Map (CMap) database. Molecular docking and molecular dynamics simulation were performed to predict the binding of the target protein to the small molecule compound, and the MM/GBSA method was used to calculate the binding free energy. Cluster analysis was performed using the cluster tool in the GROMACS package. An AAA cell-free model was built, and CETSA experiments were used to demonstrate the binding ability of small molecules to the target protein in cells. A total of 2244 DEGs in AAA were obtained through differential analysis, and the DEGs were mainly enriched in the tubulin binding biological function and cell cycle pathway. The CMap results showed that Apicidin had a potential therapeutic effect on AAA with a connectivity score of -97.74, and HDAC4 was the target protein of Apicidin. Based on literature, HDAC4-Apicidin was selected as the subsequent research object. The lowest affinity of Apicidin-HDAC4 molecular docking was -8.218kcal/mol. Molecular dynamics simulation results indicated that Apicidin-HDAC4 could form a stable complex. MM/GBSA analysis showed a total binding free energy of -55.40 ± 0.79kcal/mol, and cluster analysis showed that there were two main conformational clusters during the binding process, accounting for 22.4% and 57.8%, respectively. Apicidin could form hydrogen bonds with surrounding residues for stable binding. CETSA experiment proved the stable binding ability of Apicidin and HDAC4. Apicidin inhibited HDAC4 in AAA and exhibited favorable protein-ligand interactions and stability, making it a potential candidate drug for treating AAA.

CD8+ T-cell deficiency protects mice from abdominal aortic aneurysm formation in response to calcium chloride2.

Abdominal aortic aneurysm (AAA) is an aneurysm-like dilated and highly fatal cardiovascular disease. CD8+ T cells have been shown to be critical for vascular pathological processes, but the contribution of these lymphocytes to vascular diseases remains elusive. Eight-week-old male wildtype (CD8+/+) and Cd8a knockout (CD8-/-) mice were used in a calcium chloride2 (CaCl2)-induced experimental AAA model. At 6 weeks after surgery, CD8+ T-cell deletion prevented the formation of AAA, accompanied by reductions of the levels of inflammatory (interferon-γ [IFN-γ], interleukin-1β, monocyte chemoattractant protein-1, intracellular adhesion molecule-1, vascular cell adhesion molecule-1, NOD-like receptor protein 3, caspase-1), oxidative stress [NADPH oxidase and gp91phox], and proteolysis (cathepsin S, cathepsin K, matrix metalloproteinase-2 [MMP-2] and MMP-9) proteins and/or genes in plasma and/or AAA tissues. Immunoreactivities of MMP-2 and MMP-9 were observed in macrophages. An injection of IFN-γ and adoptive transfer of CD8+ T cells of IFN-γ+/+ mice diminished CD8-/--mediated vasculoprotective actions in the AAA mice. In vitro, IFN-γ enhanced MMP-2 and MMP-9 gelatinolytic activities in macrophage and/or vascular smooth muscle cells. The vasculoprotective effects of CD8+ T-cell deletion in a mouse CaCl2-induced AAA model were likely attributable to, at least in part, the attenuation of IFN-γ-dependent inflammation action, oxidative stress production, and proteolysis, suggesting a novel therapeutic target for AAA formation by regulating CD8+ T-cell-derived IFN-γ secretion.

Abstract Tu018: Integrin alpha 1 plays a critical role in angiotensin II-induced abdominal aortic aneurysm rupture

Background: Abdominal aortic aneurysm (AAA) is a life-threatening condition without an established specific therapy. This study aims to identify a new therapeutic target for preventing AAA rupture. Methods and Results: Angiotensin II (AngII)-infused ApoE-/- mice develop AAA, an established model of AAA. To identify molecular pathways contributing to AAA development, we conducted single-cell RNA sequence analysis using a dataset of aortic tissues harvested from mice infused with AngII for 4 weeks (GSE186865). Gene set enrichment analysis revealed upregulation of integrin- and TGFB-related pathways in AngII-stimulated smooth muscle cells (SMCs). Integrin α1 (Itga1) constitutes half of the α1β1 integrin duplex and serves as a major collagen receptor in SMCs. Male wild-type (n=37) and Itga1 knockout (n=36) mice with an ApoE-/- background received a continuous infusion of AngII for 4 weeks to develop AAA. Kaplan-Meier analysis indicated that ablating Itga1 significantly suppressed AAA rupture (p=0.009), without affecting the maximum diameter of the abdominal aorta. Considering extracellular matrix degradation as a critical hallmark of AAA rupture, we examined the abundance of major matrix metalloproteinases (MMPs) in the aortic wall, specifically MMP2 and MMP9, two major MMPs in the aorta. Gelatin zymography revealed that ablation of Itga1 suppressed AngII-induced MMP9 increase, without affecting MMP2 abundance. The expression of MMP9 is controlled by inflammation and proliferation signals; hence, we focused on these signals. qPCR analysis (n=8 per group) demonstrated that AngII-induced upregulation of Tnfa (p<0.001) and Tgfb1 (p=0.026) was mitigated by Itga1 ablation in aortic tissues. Conclusion: The integrin pathway is activated in the tissue of AAA tissue and deletion of Itga1 suppresses AAA rupture. Here we showed that the Itga1-Tgfb1-MMP9 axis would be a novel molecular target to inhibit AAA rupture.

Myeloid-Specific Thrombospondin-1 Deficiency Exacerbates Aortic Rupture via Broad Suppression of Extracellular Matrix Proteins.

Rupture of abdominal aortic aneurysms (AAA) is associated with high mortality. However, the precise molecular and cellular drivers of AAA rupture remain elusive. Our prior study showed that global and myeloid-specific deletion of matricellular protein thrombospondin-1 (TSP1) protects mice from aneurysm formation primarily by inhibiting vascular inflammation. To investigate the cellular and molecular mechanisms that drive AAA rupture by testing how TSP1 deficiency in different cell populations affects the rupture event. We deleted TSP1 in endothelial cells and macrophages --- the major TSP1-expressing cells in aneurysmal tissues ---- by crossbreeding Thbs1 flox/flox mice with VE-cadherin Cre and Lyz2-cre mice, respectively. Aortic aneurysm and rupture were induced by angiotensin II in mice with hypercholesterolemia. Myeloid-specific Thbs1 knockout, but not endothelial-specific knockout, increased the rate of lethal aortic rupture by more than 2 folds. Combined analyses of single-cell RNA sequencing and histology showed a unique cellular and molecular signature of the rupture-prone aorta that was characterized by a broad suppression in inflammation and extracellular matrix production. Visium spatial transcriptomic analysis on human AAA tissues showed a correlation between low TSP1 expression and aortic dissection. TSP1 expression by myeloid cells negatively regulates aneurysm rupture, likely through promoting the matrix repair phenotypes of vascular smooth muscle cells thereby increasing the strength of the vascular wall.

Small AAAs: Recommendations for Rodent Model Research for the Identification of Novel Therapeutics.

Most abdominal aortic aneurysms (AAAs) are small with low rupture risk (<1%/y) when diagnosed but slowly expand to ≥55 mm and undergo surgical repair. Patients and clinicians require medications to limit AAA growth and rupture, but drugs effective in animal models have not translated to patients. Use models that simulate human AAA tissue pathology, growth patterns, and rupture; focus on the clinically relevant outcomes of growth and rupture; design studies with the rigor required of human clinical trials; monitor AAA growth using reproducible ultrasound; and perform studies in both males and females. The aortic adventitial elastase oral β-aminopropionitrile model has many strengths including simulating human AAA pathology and modeling prolonged aneurysm growth. The Ang II (angiotensin II) model performed less well as it better simulates acute aortic syndrome than AAA. The elastase plus TGFβ (transforming growth factor-β) blocking antibody model displays a high rupture rate, making prolonged monitoring of AAA growth not feasible. The elastase perfusion and calcium chloride models both display limited AAA growth.

Insights on P2X7 in patients with abdominal aortic aneurysm and carotid atherosclerosis

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Ministero della Salute, Italia, Rete Cardiologica Nazionale Background Carotid atherosclerosis and abdominal aortic aneurysm (AAA) are co-morbidities [1]. P2X-purinoceptor-7 (P2X7) is expressed both in human AAA and atherosclerotic carotid plaque (CPL) and is instability-associated in CPL [2-4]. P2X7 is functionally related to miR-150 and miR-186 (two microRNA which modulate vascular cell proliferation) [5,6], and may be shed into the bloodstream in correlation with C-reactive protein increase [7]. Little is known about the possible association between AAA risk and circulating P2X7, miR-150 and miR-186 in patients with/without hemodinamically significant CPL. Purpose Our aim is exploring the possible relationship between circulating P2X7, target miRs clinical status and lesion dimensions in patients with CPL and/or AAA. Methods Male patients with AAA and/or CPL were characterized.CPL stenosis was assessed by Ecocolordoppler-TSA. Eligible cohort included 20 patients with AAA undergoing aneurysmectomy [10 with CPL stenosis ≤40% (group A), 10with CPL stenosis &amp;gt;40% (group B)], 10 patients with hemodinamically significant CPL and small or no AAA undergoing endarterectomy (group C) donating blood and vascular samples at surgery. Twelve healthy blood donors (group D) were enrolled as controls. P2X7 protein was evaluated by ELISA. P2X7 gene, miR-150 and miR-186 were determined by RT-qPCR. Results In group A, CPL appeared mainly localized at carotid bulb, in group B extended along branches. P2X7 gene was undetermined into serum and expressed without differences among groups in patient tissues. P2X7 protein was measurable in all serum samples and was lower in group B than in the others (p=0,0117, p=0,0009, p=0,0003 vs. group A, C, D, respectively) and in group C vs. D (p=0,0006). These data differentiated patients with hemodinamically significant CPLs with severe AAA from the other patients and all those with hemodinamically significant CPLs (irrespectively from AAA severity) from healthy individuals. In group B but not in the others, serum P2X7 was linearly associated to platelet concentration. In group C the amounts of P2X7 in CPL tissue and serum were negatively associated, whereas in group A and B the P2X7 levels in AAA tissue and serum were unrelated. The miRs were detected into both serum and tissue and not correlated among them nor with circulating P2X7 protein. The serum miR-186, but not miR-150, was differentially expressed in group A vs. B (p=0,0266) and linearly related to AAA diameter in group B (p=0.0351). P2X7 content and expressed gene (p=0,015, p= 0,023, respectively) and miR expressions (p=0,0418 for miR-186, p=0,0012 for miR-150) were more elevated in AAA vs. CPL tissues, highlighting the vascular heterogenicity. Conclusions Preliminary data suggest a role for P2X7 and miR-186 in profiling patients with hemodinamically significant CPL with/without high risk AAA, deserving further investigations.