Human Abdominal Aortic Aneurysm Tissue Research Articles

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.

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

Abstract 144: Pyruvate Dehydrogenase Kinase 4 Contributes To Abdominal Aortic Aneurysm Through Accelerating Smooth Muscle Cell Metabolic Reprogramming And Nlrp3 Inflammasome-dependent Pyroptosis

Background: Abdominal aortic aneurysm (AAA) is a severe aortic disease with a high mortality rate upon rupture. Understanding its molecular mechanisms of progression is crucial for therapeutic strategery. Pyruvate dehydrogenase kinase 4 (PDK4) is an important mitochondrial matrix enzyme in cellular metabolism. This study explored the role of PDK4 in AAA development, focusing on its impact on vascular smooth muscle cells (VSMCs). Methods: The expression of PDK4 in human and mice AAA tissues was measured. The role of PDK4 in AAA development was evaluated in VSMC-specific PDK4 knockout mice using a PCSK9/Ang II-induced AAA model. Seahorse analysis was used to assess oxidative phosphorylation and glycolysis levels. RNA-sequencing analysis and mitochondrial functional analysis were used to explore the molecular mechanisms. Results: We observed an increased expression of VSMC-PDK4 in human AAA tissues and AAA tissues from a PCSK9/AngII-induced AAA mouse model. PDK4 deficiency in VSMCs significantly inhibited AAA formation, whereas PDK4 overexpression aggravated AAA development. PDK4 promoted the metabolic reprogramming of VSMCs and VSMC-specific deletion of PDK4 ameliorated the contractile-to-synthetic phenotype switch in VSMCs. PDK4 activated NLRP3 inflammasome by impairing mitochondrial function, which contributed to the pyroptosis of VSMCs and instigated an inflammatory response. Mitochondria-targeted ROS scavenging and NLRP3 inhibitor MCC950 effectively attenuated NLRP3 inflammasome activation and pyroptosis mediated by PDK4. Supplementation with MCC950 was beneficial to ameliorating AAA in a VSMC PDK4-dependent manner in mouse models. Conclusion: PDK4 promotes smooth muscle cell metabolic reprogramming and NLRP3 inflammasome-dependent pyroptosis in AAA development, suggesting that targeting PDK4 may be a promising therapeutic strategy for the prevention and treatment of AAA.

Abstract 3010: Inhibition Of Endothelial Cell-Mediated Efferocytosis Promotes Abdominal Aortic Aneurysm Formation

Introduction: Aortic wall inflammation and vascular smooth muscle (VSMC) apoptosis can lead to accumulation of apoptotic cells and debris during abdominal aortic aneurysm (AAA) formation. We hypothesize that dysregulation of MerTK receptor-mediated efferocytosis (clearance of apoptotic cell debris) by endothelial cells (ECs) can significantly contribute to aortic inflammation and vascular remodeling in AAA pathogenesis. Methods: Single-cell RNA sequencing of human AAA tissue from a published dataset (PMID: 33779682) was evaluated for cells of the aortic wall (VSMC/EC) identified via SingleR and cell-specific marker s. Differentially expressed genes (DEGs) were identified via edgeR using the QL F-test and efferocytosis-related genes (ERGs) were recognized using a list of 141 ERGs curated from GeneCards. MerTK expression was assessed in human aortic tissue and ECs via western blot and immunofluorescence. PKH67-labelled apoptotic VSMCs were overlayed on ECs to assess efferocytosis activity and visualized via confocal microscopy. In a murine elastase AAA model, endothelial-specific MerTK knockout ( Cdh5 Cre-ERT2 / MerTK fl/fl ) and littermate male mice were analyzed on postoperative day 14 for aortic diameter. Statistical analyses were performed by Welch’s T-test and p&lt;0.05 was considered statistically significant. Results: ERGs (ANXA2, PHACTR1, PPARG, ADAM9, CD14, IL6) were differentially expressed (p&lt;0.05, log 2 FC &gt; |1|) between aortic wall cells of human AAAs and control. Human AAA tissue displayed a significant increase in MerTK protein compared to controls (1±0.1 vs. 1.51±0.4, n=6/group, p=0.01). The expression of MerTK in aortic ECs was confirmed by western blot and immunofluorescence(n=3). Pharmacologic inhibition of MerTK using UNC5293, a specific MerTK inhibitor, attenuated EC-mediated efferocytosis of apoptotic VSMCs (qualitative data; 10 images/group n=4). Importantly, EC-specific MerTK -/- mice had a significant increase in aortic diameter compared to littermate controls (155±40.4 vs. 103±30.9%, n=5-6/group, p=0.04) on day 14. Conclusion: EC-mediated efferocytosis can be regulated by MerTK to mediate AAA formation. Ongoing studies will delineate mechanisms of EC-MerTK signaling in AAA formation.

Abstract 1006: Pathological PERK/ATF4 Activation In Vascular Smooth Muscle Cells Drives Abdominal Aortic Aneurysm Development

Introduction: Abdominal aortic aneurysms (AAA) are characterized by vascular smooth muscle cell (VSMC) apoptosis leading to pathological aortic remodeling. Activation of the endoplasmic reticulum (ER) stress response has been linked to VSMC dysfunction but the driving factors behind ER stress activation and its role on AAA development remains poorly defined. The epigenetic enzyme, MLL1, plays a critical role in establishing cellular phenotype and regulating gene expression. We examine the MLL-mediated epigenetic regulation of the ER stress response in VSMCs during AAA development and establish the ER stress pathways as a novel target in the treatment AAAs. Methods: Single-cell sequencing was conducted on human AAA and control tissue samples. For our murine model, C57BL/6 mice were injected with an AAV encoding a PCSK9 gain-of-function mutation and then fed a saturated fat-enriched diet and infused with AngII (1 μg/kg/min) with or without 4-PBA (20mg/kg/day). Mice with vascular smooth muscle cell-specific knockout of PERK (PERK SMKO ) underwent tamoxifen injection. AAA maximum diameters were quantified and VSMCs were isolated. Eif2a, Atf4 , and Chop gene expression was examined. Apoptosis was quantified by Annexin V staining. Results: Using single-cell RNA sequencing of human aortic tissue, we identified EIF2A, ATF4 , and CHOP were profoundly upregulated in aneurysmal VSMCs as well as two murine AAA models. Mechanistically, TNFα signaling in VSMCs leads to activation of MLL1 which trimethylates histone 3 lysine 4 (H3K4me3) at NF-κB binding sites on the EIF2A and ATF4 gene promoters thereby upregulating the ER stress response and driving VSMC apoptosis. Both In vivo pharmacological inhibition of the PERK/eIF2A/ATF4 ER stress response via 4-PBA injection and vascular smooth muscle depletion of PERK (PERK SMKO ) inhibited AAA formation resulting with in significant reduction in VSMC apoptosis, elastin fragmentation, and inflammatory cytokine infiltration. Conclusions: Taken together, our results identify MLL1-mediated activation of the PERK/eIF2A/ATF4 ER stress response as a critical regulator of VSMC apoptosis in AAA formation and suggest inhibition of ER stress response as a mechanistic target for the management of AAAs.

Terazosin attenuates abdominal aortic aneurysm formation by downregulating Peg3 expression to inhibit vascular smooth muscle cell apoptosis and senescence

Abdominal aortic aneurysm (AAA), a vascular degenerative disease, is a potentially life-threatening condition characterised by the loss of vascular smooth muscle cells (VSMCs), degradation of extracellular matrix (ECM), inflammation, and oxidative stress. Despite the severity of AAA, effective drugs for treatment are scarce. At low doses, terazosin (TZ) exerts antiapoptotic and anti-inflammatory effects in several diseases, but its potential to protect against AAA remains unexplored. Herein, we investigated the effects of TZ in two AAA animal models: Angiotensin II (Ang II) infusion in Apoe−/− mice and calcium chloride application in C57BL/6J mice. Mice were orally administered with TZ (100 or 1000 μg/kg/day). The in vivo results indicated that low-dose TZ alleviated AAA formation in both models. Low-dose TZ significantly reduced aortic pulse wave velocity without exerting an apparent antihypertensive effect in the Ang II-induced AAA model. Paternally expressed gene 3 (Peg3) was identified via RNA sequencing as a novel TZ target. PEG3 expression was significantly elevated in both mouse and human AAA tissues. TZ suppressed PEG3 expression and reduced the abundance of matrix metalloproteinases (MMP2/MMP9) in the tunica media. Functional experiments and molecular analyses revealed that TZ (10 nM) treatment and Peg3 knockdown effectively prevented Ang II-induced VSMC senescence and apoptosis in vitro. Thus, Peg3, a novel target of TZ, mediates inflammation-induced VSMC apoptosis and senescence. Low-dose TZ downregulates Peg3 expression to attenuate AAA formation and ECM degradation, suggesting a promising therapeutic strategy for AAA.

Ganglioside GM3 Protects Against Abdominal Aortic Aneurysm by Suppressing Ferroptosis.

Abdominal aortic aneurysm (AAA) is a potentially life-threatening vascular condition, but approved medical therapies to prevent AAA progression and rupture are currently lacking. Sphingolipid metabolism disorders are associated with the occurrence and development of AAA. It has been discovered that ganglioside GM3, a sialic acid-containing type of glycosphingolipid, plays a protective role in atherosclerosis, which is an important risk factor for AAA; however, the potential contribution of GM3 to AAA development has not been investigated. We performed a metabolomics study to evaluated GM3 level in plasma of human patients with AAA. We profiled GM3 synthase (ST3GAL5) expression in the mouse model of aneurysm and human AAA tissues through Western blotting and immunofluorescence staining. RNA sequencing, affinity purification and mass spectrometry, proteomic analysis, surface plasmon resonance analysis, and functional studies were used to dissect the molecular mechanism of GM3-regulating ferroptosis. We conditionally deleted and overexpressed St3gal5 in smooth muscle cells (SMCs) in vivo to investigate its role in AAA. We found significantly reduced plasma levels of GM3 in human patients with AAA. GM3 content and ST3GAL5 expression were decreased in abdominal aortic vascular SMCs in patients with AAA and an AAA mouse model. RNA sequencing analysis showed that ST3GAL5 silencing in human aortic SMCs induced ferroptosis. We showed that GM3 interacted directly with the extracellular domain of TFR1 (transferrin receptor 1), a cell membrane protein critical for cellular iron uptake, and disrupted its interaction with holo-transferrin. SMC-specific St3gal5 knockout exacerbated iron accumulation at lesion sites and significantly promoted AAA development in mice, whereas GM3 supplementation suppressed lipid peroxidation, reduced iron deposition in aortic vascular SMCs, and markedly decreased AAA incidence. Together, these results suggest that GM3 dysregulation promotes ferroptosis of vascular SMCs in AAA. Furthermore, GM3 may constitute a new therapeutic target for AAA.

Abstract 12885: Pathological Perk/Atf4 Activation in Abdominal Aortic Aneurysm Formation

Introduction: Abdominal aortic aneurysms (AAA) are characterized by vascular smooth muscle cell (VSMC) apoptosis leading to pathological aortic remodeling. Activation of the endoplasmic reticulum (ER) stress response has been linked to VSMC dysfunction but the driving factors behind ER stress activation and its role on AAA development remains poorly defined. The epigenetic enzyme, MLL1, plays a critical role in establishing cellular phenotype and regulating gene expression. We examine the MLL-mediated epigenetic regulation of the ER stress response in VSMCs during AAA development and establish the ER stress pathways as a novel target in the treatment AAAs. Methods: Single-cell sequencing was conducted on human AAA and control tissue samples. For our murine model, C57BL/6 mice were injected with an AAV encoding a PCSK9 gain-of-function mutation and then fed a saturated fat-enriched diet and infused with AngII (1 μg/kg/min) with or without 4-PBA (20mg/kg/day). The elastase induced AAA model was used as a secondary murine model. AAA maximum diameters were quantified and VSCMs were isolated. Eif2a, Atf4 , and Chop gene expression was examined. Apoptosis was quantified by Annexin V staining. Results: Using single-cell RNA sequencing of human aortic tissue, we identified EIF2A, ATF4 , and CHOP were profoundly upregulated in aneurysmal VSMCs as well as two murine AAA models. Mechanistically, TNFα signaling in VSMCs leads to activation of MLL1 which trimethylates histone 3 lysine 4 (H3K4me3) at NF-κB binding sites on the EIF2A and ATF4 gene promoters thereby upregulating the ER stress response and driving VSMC apoptosis. In vivo pharmacological inhibition of the eIF2A/ATF4 ER stress response via 4-PBA injection inhibited AAA formation in both the AngII-induced and elastase-induced AAA model resulting with in significant reduction in VSMC apoptosis, elastin fragmentation, and inflammatory cytokine infiltration. Conclusions: Taken together, our results identify MLL1-mediated activation of the eIF2A/ATF4 ER stress response as a critical regulator of VSMC apoptosis in AAA formation and suggest inhibition of ER stress response as a mechanistic target for the management of AAAs.

Curcumin mediates macrophage polarization to inhibit the formation of abdominal aortic aneurysms by inhibiting the expression of histone acetyltransferase EP300

BackgroundThe onset of abdominal aortic aneurysm (AAA) is caused by local dilatation of the infrarenal abdominal aorta. Curcumin can affect the polarization and function of macrophage subsets in the context of vascular diseases. Curcumin, a natural EP300 inhibitor, may have therapeutic effects on vascular diseases. ObjectiveTo explore the therapeutic pathway of curcumin in AAA. MethodsWe found that the hub regulatory factor EP300 is the key factor by which curcumin acts on AAA through bioinformatics network pharmacology and molecular docking. We verified the localization of histone acetyltransferase EP300 in AAA tissue by immunohistochemistry (IHC) and immunofluorescence (IF) staining. Then, we used Real-time polymerase chain reaction (RT-PCR) to explore the high expression level of EP300 in human AAA tissues and analysed its correlation with key genes in AAA diseases. In addition, we successfully induced M1 macrophages and EP300 knockout macrophages with LPS/IFN-γ in vitro to investigate the effect of EP300 on macrophage polarization. ResultsIn our study, thirty important targets, including AKT1, STAT3, EGFR, EP300, GSK3B, APP, SERPINE1 and MMP14, were involved in the interaction between AAA and curcumin. Our molecular docking results showed that EP300 spontaneously binds to curcumin most easily. Therefore, we subsequently identified the histone acetyltransferase EP300 as the hub target of curcumin in AAA. IHC and IF staining of the outer membrane of AAA showed that EP300 was located in the mesa of AAA tissue, which was also the place where macrophages gathered. The mRNA expression of EP300 in human AAA tissue was upregulated, as shown by RT-PCR (P = 0.01). In in vitro RAW264.7 cells, we found that EP300 knockout in M0 macrophages significantly promoted the anti-inflammatory M2 polarization of macrophages and inhibited the proinflammatory M1 polarization of macrophages. ConclusionOur research showed that in the therapeutic mechanism by which curcumin treats human AAA, histone acetyltransferase EP300 affected the progression of AAA by promoting aortic inflammation, acting on the immune environment, modulating macrophage proinflammatory polarization, and influencing the expression of other AAA target genes.

The Histone Methyltransferase SETDB2 Modulates Tissue Inhibitors of Metalloproteinase-Matrix Metalloproteinase Activity During Abdominal Aortic Aneurysm Development.

To determine macrophage-specific alterations in epigenetic enzyme function contributing to the development of abdominal aortic aneurysms (AAAs). AAA is a life-threatening disease, characterized by pathologic vascular remodeling driven by an imbalance of matrix metalloproteinases and tissue inhibitors of metalloproteinases (TIMPs). Identifying mechanisms regulating macrophage-mediated extracellular matrix degradation is of critical importance to developing novel therapies. The role of SET Domain Bifurcated Histone Lysine Methyltransferase 2 (SETDB2) in AAA formation was examined in human aortic tissue samples by single-cell RNA sequencing and in a myeloid-specific SETDB2 deficient murine model induced by challenging mice with a combination of a high-fat diet and angiotensin II. Single-cell RNA sequencing of human AAA tissues identified SETDB2 was upregulated in aortic monocyte/macrophages and murine AAA models compared with controls. Mechanistically, interferon-β regulates SETDB2 expression through Janus kinase/signal transducer and activator of transcription signaling, which trimethylates histone 3 lysine 9 on the TIMP1-3 gene promoters thereby suppressing TIMP1-3 transcription and leading to unregulated matrix metalloproteinase activity. Macrophage-specific knockout of SETDB2 ( Setdb2f/fLyz2Cre+ ) protected mice from AAA formation with suppression of vascular inflammation, macrophage infiltration, and elastin fragmentation. Genetic depletion of SETDB2 prevented AAA development due to the removal of the repressive histone 3 lysine 9 trimethylation mark on the TIMP1-3 gene promoter resulting in increased TIMP expression, decreased protease activity, and preserved aortic architecture. Lastly, inhibition of the Janus kinase/signal transducer and activator of the transcription pathway with an FDA-approved inhibitor, Tofacitinib, limited SETDB2 expression in aortic macrophages. These findings identify SETDB2 as a critical regulator of macrophage-mediated protease activity in AAAs and identify SETDB2 as a mechanistic target for the management of AAAs.

Down-regulation of the Smad signaling by circZBTB46 via the Smad2-PDLIM5 axis to inhibit type I collagen expression.

Abnormal type I collagen (COL1) expression is associated with the development of many cardiovascular diseases. The TGF-beta/Smad signaling pathway and circRNAs have been shown to regulate COL1 gene expression, but the underlying molecular mechanisms are still not fully understood. Gain- and loss-of-function experiments were prformed to study the effect of circZBTB46 on the expression of alpha 2 chain of type I collagen (COL1A2). Co-immunoprecipitation assay was performed to observe the interaction between two proteins. RNA immunoprecipitation assay and biotin pull-down assay were performed to observe the interaction of circZBTB46 with PDLIM5. In this study, we investigated the role of circZBTB46 in regulating COL1A2 expression in human vascular smooth muscle cells (VSMCs). We found that circZBTB46 is expressed in VSMCs and that TGF-beta inhibits circZBTB46 formation by downregulating KLF4 expression through activation of the Smad signaling pathway. CircZBTB46 inhibits the expression of COL1A2 induced by TGF-beta. Mechanistically, circZBTB46 mediates the interaction between Smad2 and PDLIM5, resulting in the inhibition of Smad signaling and the subsequent downregulation of COL1A2 expression. Furthermore, we found that the expression of TGF-beta and COL1A2 is decreased, while circZBTB46 expression is increased in human abdominal aortic aneurysm tissues, indicating that circZBTB46-mediated regulation of TGF-beta/Smad signaling and COL1A2 synthesis in VSMCs plays a crucial role in vascular homeostasis and aneurysm development. CircZBTB46 was identified as a novel inhibitor of COL1 synthesis in VSMCs, highlighting the importance of circZBTB46 and PDLIM5 in regulating TGF-beta/Smad signaling and COL1A2 expression.

Abstract 282: Upregulation Of Receptor Interacting Protein Kinase-3 Augments Abdominal Aortic Aneurysms

Objective: Aortic smooth muscle cell (SMC) depletion is a major pathological feature of abdominal aortic aneurysm (AAA). Our lab has previously reported elevated levels of Receptor Interacting Protein Kinase-3 (RIPK3), a major mediator of necroptosis, in human AAA tissues. Pharmacologic inhibition of RIPK3 or deletion of the Ripk3 gene prevents aortic SMC death and attenuates aneurysm growth in murine models. In the current study, we tested the hypothesis that manipulation of Ripk3 gene expression would alter AAA development. Methods: We manipulated Ripk3 gene expression by mutating a conserved putative transcription enhancer site using CRISPR-Cas9-mediated gene editing. AAA was induced in 8-week old male CRISPR mutated (CM) and wild-type (WT) mice by perivascular application of CaCl 2 or NaCl (sham). Aortic tissues were harvested on days 4 or 28 post-surgery and analyzed by immunohistochemistry (IHC). Primary aortic SMCs isolated from both groups were stimulated with inflammation-related cytokines and Ripk3 mRNA was examined by RT-PCR. Results: Both CM and WT mice developed AAA in response to CaCl 2 . Change in aortic diameter was greater in CM mice than WT at 28 days (66.81±4.512 vs 44.87±6.685%, P =0.0108). IHC showed CM aneurysm tissue to exhibit fewer SMCs and a greater number of non-SMC cells in the tunica media, presumably infiltrated immune cells. Furthermore, mutant tissues contained higher levels of RIPK3 positivity than WT aortas. Consistently, CM aortic SMCs expressed more Ripk3 mRNA than WT SMCs, both with vehicle and following stimulation with IL-1β, IL-25, and IL-33. Conclusions: Our data show a positive correlation between Ripk3 expression and aneurysm size. We are currently analyzing data from experiments aimed to prove the causal relationship between Ripk3 gene editing, SMC necroptosis, inflammation, and aneurysm growth. This study further illustrates the potential of RIPK3 as a therapeutic target of AAA.

Mouse Abdominal Aortic Aneurysm Model Induced by Periarterial Incubation of Papain

For decades, numerous experimental animal models have been developed to examine the pathophysiologic mechanisms and potential treatments for abdominal aortic aneurysms (AAAs) in diverse species with varying chemical or surgical approaches. This study aimed to create an AAA mouse model by the periarterial incubation with papain, which can mimic human AAA with advantages such as simplicity, convenience, and high efficiency. Eighty C57BL/6J male mice were randomly assigned to 1 of the 4 groups: papain (1.0 or 2.0 mg), porcine pancreatic elastase, and phosphate-buffered solution. The aortic segment was wrapped for 20 minutes, and the diameter was measured using ultrasound preoperatively and postoperative days 7 and 14. Then, the mice were killed for histomorphometric and immunohistochemical analyses. According to ultrasound measurements and histomorphometric analyses, on postoperative day 7, 65% of mice in the 1.0-mg papain group and 60% of mice in the 2.0-mg papain group developed AAA. In both papain groups, 100% of mice developed AAA, and 65% of mice in the porcine pancreatic elastase group developed AAA on postoperative day 14. Furthermore, hematoxylin/eosin, elastin van Gieson, and Masson staining of tissues from the papain group revealed thickened media and intimal hyperplasia, collagen sediments, and elastin destruction, indicating that AAA histochemical alteration was similar to that of humans. In addition, the immunohistochemical analysis was conducted to detect infiltrated inflammatory cells, such as macrophages and leukocytes, in the aortic wall and hyperplasic adventitia. The expression of matrix metalloproteinase 2 and 9 was significantly upregulated in papain and human AAA tissues. Periarterial incubation with 1.0 mg of papain for 20 minutes can successfully create an experimental AAA model in mice for 14 days, which can be used to explore the mechanism and treatment of human AAA.

Revealing the Intercellular Communication Changes of Human Abdominal Aortic Aneurysms at Single-cell Resolution

Abstract Background: Abdominal aortic aneurysm (AAA) is a chronic vascular inflammation progression with a high mortality. However, intercellular communication has not been sufficiently explored in human AAA tissue. Aims: Here, we investigated available single-cell RNA sequencing data generated from human AAA. Results: These cells were divided into 27 clusters and 6 main cell types, such as T-cells, B-cells, myeloid cells, neutrophils, endothelial cells, and fibroblasts. Immune cells were present in AAA tissues more than control. In addition, we established an intercellular communication network and observed a more pronounced enrichment of TGFB1, CCL, VEGF, IL6, and complement pathways in the AAA group compared to the control group. Conclusion: Together, our analyses provide beneficial insight into cell–cell communication and expand channels for the effective immunotherapy development of AAA in the future.

Reactive oxygen species-induced long intergenic noncoding RNA p21 accelerates abdominal aortic aneurysm formation by promoting secretary smooth muscle cell phenotypes

Whether long noncoding RNAs participate in the formation of abdominal aortic aneurysms (AAAs) through the regulation of SMC phenotypic switching is unknown. lincRNA-p21 induced by reactive oxygen species (ROS) is likely functionally associated with SMC phenotypic switching. We thus investigated the role of lincRNA-p21 in SMC phenotypic switching-associated AAA formation and its underlying mechanisms. An analysis of human and mouse abdominal aortic samples revealed that the lincRNA-p21 levels were significantly higher in AAA tissue. Stimulation with hydrogen peroxide upregulated the expression of lincRNA-p21 in a dose-dependent manner and converted SMCs from a contractile phenotype to a synthetic, proteolytic, and proinflammatory phenotype in vitro. Moreover, lincRNA-p21 promoted fracture of elastic fibres, reconstruction of the vascular wall, and AAA formation in vivo by modulating SMC phenotypic switching in two mouse models of AAA induced by angiotensin II or porcine pancreatic elastase (PPE) perfusion. Using a bioinformatics prediction method and luciferase reporter gene assays, we further proved that lincRNA-p21 sponged miR-204-5p to release the transcriptional activity of Mekk3 and promoted the NF-κB pathway and thereby played a role in the SMC phenotypic switch and AAA formation. The ROS levels were positively correlated with the lincRNA-p21 levels in human and mouse AAA tissues. The knockdown of lincRNA-p21 in a PPE-induced mouse AAA model increased the miR-204-5p levels and reduced the expression of Mekk3, whereas lincRNA-p21 overexpression had the opposite effect. Collectively, the results indicated that ROS-induced lincRNA-p21 sponges miR-204-5p to accelerate synthetic and proinflammatory SMC phenotypes through the Mekk3/NF-κB pathway in AAA formation. Thus, lincRNA-p21 may have therapeutic potential for AAA formation.

BAF60c prevents abdominal aortic aneurysm formation through epigenetic control of vascular smooth muscle cell homeostasis.

Abdominal aortic aneurysm (AAA) is a life-threatening vascular disease. BAF60c, a unique subunit of the SWItch/sucrose nonfermentable (SWI/SNF) chromatin remodeling complex, is critical for cardiac and skeletal myogenesis, yet little is known about its function in the vasculature and, specifically, in AAA pathogenesis. Here, we found that BAF60c was downregulated in human and mouse AAA tissues, with primary staining to vascular smooth muscle cells (VSMCs), confirmed by single-cell RNA-sequencing. In vivo studies revealed that VSMC-specific knockout of Baf60c significantly aggravated both angiotensin II- (Ang II-) and elastase-induced AAA formation in mice, with a significant increase in elastin degradation, inflammatory cell infiltration, VSMC phenotypic switch, and apoptosis. In vitro studies showed that BAF60c knockdown in VSMCs resulted in loss of contractile phenotype, increased VSMC inflammation, and apoptosis. Mechanistically, we demonstrated that BAF60c preserved VSMC contractile phenotype by strengthening serum response factor (SRF) association with its coactivator P300 and the SWI/SNF complex and suppressing VSMC inflammation by promoting a repressive chromatin state of NF-κB target genes as well as preventing VSMC apoptosis through transcriptional activation of KLF5-dependent B cell lymphoma 2 (BCL2) expression. Our identification of the essential role of BAF60c in preserving VSMC homeostasis expands its therapeutic potential in preventing and treating AAA.

Resolution of inflammation via RvD1/FPR2 signaling mitigates Nox2 activation and ferroptosis of macrophages in experimental abdominal aortic aneurysms.

Abdominal aortic aneurysm (AAA) formation is characterized by inflammation, leukocyte infiltration, and vascular remodeling. Resolvin D1 (RvD1) is derived from ω-3 polyunsaturated fatty acids and is involved in the resolution phase of chronic inflammatory diseases. The aim of this study was to decipher the protective role of RvD1 via formyl peptide receptor 2 (FPR2) receptor signaling in attenuating abdominal aortic aneurysms (AAA). The elastase-treatment model of AAA in C57BL/6 (WT) mice and human AAA tissue was used to confirm our hypotheses. Elastase-treated FPR2-/- mice had a significant increase in aortic diameter, proinflammatory cytokine production, immune cell infiltration (macrophages and neutrophils), elastic fiber disruption, and decrease in smooth muscle cell α-actin expression compared to elastase-treated WT mice. RvD1 treatment attenuated AAA formation, aortic inflammation, and vascular remodeling in WT mice, but not in FPR2-/- mice. Importantly, human AAA tissue demonstrated significantly decreased FPR2 mRNA expression compared to non-aneurysm human aortas. Mechanistically, RvD1/FPR2 signaling mitigated p47phox phosphorylation and prevented hallmarks of ferroptosis, such as lipid peroxidation and Nrf2 translocation, thereby attenuating HMGB1 secretion. Collectively, this study demonstrates RvD1-mediated immunomodulation of FPR2 signaling on macrophages to mitigate ferroptosis and HMGB1 release, leading to resolution of aortic inflammation and remodeling during AAA pathogenesis.

Potential of Disease-Modifying Anti-Rheumatic Drugs to Limit Abdominal Aortic Aneurysm Growth.

Inflammation is strongly implicated in the pathogenesis of abdominal aortic aneurysms (AAA). This review examined the potential role of biologic disease-modifying anti-rheumatic drugs (bDMARDs) as repurposed drugs for treating AAA. Published evidence from clinical and preclinical studies was examined. Findings from animal models suggested that a deficiency or inhibition of tumour necrosis factor-α (TNF-α) (standard mean difference (SMD): −8.37, 95% confidence interval (CI): −9.92, −6.82), interleukin (IL)-6 (SMD: −1.44, 95% CI: −2.85, −0.04) and IL-17 (SMD: −3.36, 95% CI: −4.21, −2.50) led to a significantly smaller AAA diameter compared to controls. Human AAA tissue samples had significantly increased TNF-α (SMD: 1.68, 95% CI: 0.87, 2.49), IL-1β (SMD: 1.93, 95% CI: 1.08, 2.79), IL-6 (SMD: 2.56, 95% CI: 1.79, 3.33) and IL-17 (SMD: 6.28, 95% CI: 3.57, 8.99) levels compared to non-AAA controls. In human serum, TNF-α (SMD: 1.11, 95% CI: 0.25, 1.97) and IL-6 (SMD: 1.42, 95% CI: 0.91, 1.92) levels were significantly elevated compared to non-AAA controls. These findings implicate TNF-α, IL-17 and IL-6 in AAA pathogenesis. Randomised controlled trials testing the value of bDMARDs in limiting AAA growth may be warranted.

Phosphodiesterase 4D contributes to angiotensin II-induced abdominal aortic aneurysm through smooth muscle cell apoptosis

Abdominal aortic aneurysm (AAA) is a permanent expansion of the abdominal aorta that has a high mortality but limited treatment options. Phosphodiesterase (PDE) 4 family members are cAMP-specific hydrolyzing enzymes and have four isoforms (PDE4A-PDE4D). Several pan-PDE4 inhibitors are used clinically. However, the regulation and function of PDE4 in AAA remain largely unknown. Herein, we showed that PDE4D expression is upregulated in human and angiotensin II-induced mouse AAA tissues using RT-PCR, western blotting, and immunohistochemical staining. Furthermore, smooth muscle cell (SMC)-specific Pde4d knockout mice showed significantly reduced vascular destabilization and AAA development in an experimental AAA model. The PDE4 inhibitor rolipram also suppressed vascular pathogenesis and AAA formation in mice. In addition, PDE4D deficiency inhibited caspase 3 cleavage and SMC apoptosis in vivo and in vitro, as shown by bulk RNA-seq, western blotting, flow cytometry and TUNEL staining. Mechanistic studies revealed that PDE4D promotes apoptosis by suppressing the activation of cAMP-activated protein kinase A (PKA) instead of the exchange protein directly activated by cAMP (Epac). Additionally, the phosphorylation of BCL2-antagonist of cell death (Bad) was reversed by PDE4D siRNA in vitro, which indicates that PDE4D regulates SMC apoptosis via the cAMP-PKA-pBad axis. Overall, these findings indicate that PDE4D upregulation in SMCs plays a causative role in AAA development and suggest that pharmacological inhibition of PDE4 may represent a potential therapeutic strategy.

Single-cell Transcriptomics Reveals Dynamic Role of Smooth Muscle Cells and Enrichment of Immune Cell Subsets in Human Abdominal Aortic Aneurysms.

To determine cell-specific gene expression profiles that contribute to development of abdominal aortic aneurysms (AAAs). AAAs represent the most common pathological aortic dilation leading to the fatal consequence of aortic rupture. Both immune and structural cells contribute to aortic degeneration, however, gene specific alterations in these cellular subsets are poorly understood. We performed single-cell RNA sequencing (scRNA-seq) analysis of AAAs and control tissues. AAA-related changes were examined by comparing gene expression profiles as well as detailed receptor-ligand interactions. An integrative analysis of scRNA-seq data with large genome-wide association study data was conducted to identify genes critical for AAA development. Using scRNA-seq we provide the first comprehensive characterization of the cellular landscape in human AAA tissues. Unbiased clustering analysis of transcriptional profiles identified seventeen clusters representing 8 cell lineages. For immune cells, clustering analysis identified 4 T-cell and 5 monocyte/macrophage subpopulations, with distinct transcriptional profiles in AAAs compared to controls. Gene enrichment analysis on immune subsets identified multiple pathways only expressed in AAA tissue, including those involved in mitochondrial dysfunction, proliferation, and cytokine secretion. Moreover, receptor-ligand analysis defined robust interactions between vascular smooth muscle cells and myeloid populations in AAA tissues. Lastly, integrated analysis of scRNA-seq data with genome-wide association study studies determined that vascular smooth muscle cell expression of SORT1 is critical for maintaining normal aortic wall function. Here we provide the first comprehensive evaluation of single-cell composition of the abdominal aortic wall and reveal how the gene expression landscape is altered in human AAAs.