Immune Response Associated Gene Signatures in Aortic Dissection Compared to Aortic Aneurysm.

BACKGROUND: Thoracic aortic dissections (TAD) are life-threatening events mostly requiring immediate surgical treatment. Although dissections mainly occur independently of thoracic aortic aneurysms (TAA), both share a high comorbidity. There are several indications for an involvement of the immune system in the development of TAD, just as in TAA. Nevertheless, specific disease-relevant genes, biomolecular processes, and immune-specific phenotypes remain unknown. METHODS: RNA from isolated aortic smooth muscle cells from TAD (n = 4), TAA (n = 3), and control patients were analyzed using microarray-based technologies. Additionally, three publicly available bulk RNA-seq studies of TAD (n = 23) and controls (n = 17) and one single-cell RNA-seq study of TAA (n = 8) and controls (n = 3) were analyzed. Differentially expressed genes were identified and used to identify affected pathways in TAD. Five selected genes were validated by quantitative real-time polymerase chain reaction (PCR). RESULTS: We identified 37 genes that were significantly dysregulated in at least three TAD studies-24 of them were not shown to be associated with TAD, yet. Gene ontology analysis showed that immune response was significantly affected. Five of the genes (CCL2, RNASE2, HAVCR2, CXCL8, and IL6R) were revealed as core genes that affect immune response in TAD. We compared the gene expression of those genes to TAA and found that CXCL8, IL6R, and potentially also CCL2 were upregulated in TAD. CONCLUSIONS: The identified immune-related genes showed TAD-specificity, independent of possible pre-existing comorbidities like TAA. So, these genes represent potential biomarkers and therapeutic targets linked to the immune response in acute TAD. Additionally, we identified a set of differentially expressed genes that represents a resource for further studies.

Versican accumulation drives Nos2 induction and aortic disease in Marfan syndrome via Akt activation.

Thoracic aortic aneurysm and dissection (TAAD) is a life-threatening condition associated with Marfan syndrome (MFS), a disease caused by fibrillin-1 gene mutations. While various conditions causing TAAD exhibit aortic accumulation of the proteoglycans versican (Vcan) and aggrecan (Acan), it is unclear whether these ECM proteins are involved in aortic disease. Here, we find that Vcan, but not Acan, accumulated in Fbn1C1041G/+ aortas, a mouse model of MFS. Vcan haploinsufficiency protected MFS mice against aortic dilation, and its silencing reverted aortic disease by reducing Nos2 protein expression. Our results suggest that Acan is not an essential contributor to MFS aortopathy. We further demonstrate that Vcan triggers Akt activation and that pharmacological Akt pathway inhibition rapidly regresses aortic dilation and Nos2 expression in MFS mice. Analysis of aortic tissue from MFS human patients revealed accumulation of VCAN and elevated pAKT-S473 staining. Together, these findings reveal that Vcan plays a causative role in MFS aortic disease in vivo by inducing Nos2 via Akt activation and identify Akt signaling pathway components as candidate therapeutic targets.

Murine double minute 2-mediated estrogen receptor 1 degradation activates macrophage migration inhibitory factor to promote vascular smooth muscle cell dedifferentiation and oxidative stress during thoracic aortic aneurysm progression.

Estrogen receptor 1 (ESR1) has been recently demonstrated as a potential diagnostic biomarker for thoracic aortic aneurysm (TAA). However, its precise role in the progression of TAA remains unclear. In this study, TAA models were established in ApoE-knockout mice and primary mouse vascular smooth muscle cells (VSMCs) through treatment with angiotensin (Ang) II. Our findings revealed a downregulation of ESR1 in Ang II-induced TAA mice and VSMCs. Upregulation of ESR1 mitigated expansion and cell apoptosis in the mouse aorta, reduced pathogenetic transformation of VSMCs, and reduced inflammatory infiltration and oxidative stress both in vitro and in vivo. Furthermore, we identified macrophage migration inhibitory factor (MIF) as a biological target of ESR1. ESR1 bound to the MIF promoter to suppress its transcription. Artificial MIF restoration negated the mitigating effects of ESR1 on TAA. Additionally, we discovered that murine double minute 2 (MDM2) was highly expressed in TAA models and mediated protein degradation of ESR1 through ubiquitination modification. Silencing of MDM2 reduced VSMC dedifferentiation and suppressed oxidative stress. However, these effects were reversed upon further silencing of ESR1. In conclusion, this study demonstrates that MDM2 activates MIF by mediating ESR1 degradation, thus promoting VSMC dedifferentiation and oxidative stress during TAA progression.

Nerve growth factor (Ngf) gene-driven semaphorin 3a (Sema3a) expression exacerbates thoracic aortic aneurysm dissection in mice.

Thoracic aortic aneurysm and dissection (TAAD) is a life-threatening disease and currently there is no pharmacological therapy. Sympathetic nerve overactivity plays an important role in the development of TAAD. Sympathetic innervation is mainly controlled by nerve growth factor (NGF, a key neural chemoattractant) and semaphoring 3A (Sema3A, a key neural chemorepellent), while the roles of these two factors in aortic sympathetic innervation and especially TAAD are unknown. We hypothesized that genetically manipulating the NGF/Sema3A ratio by the Ngf -driven Sema3a expression approach may reduce aortic sympathetic nerve innervation and mitigate TAAD progression. A mouse strain of Ngf gene-driven Sema3a expression (namely NgfSema3a/Sema3a mouse) was established by inserting the 2A-Sema3A expression frame to the Ngf terminating codon using CRISPR/Cas9 technology. TAAD was induced by ß-aminopropionitrile monofumarate (BAPN) both in NgfSema3a/Sema3a mice and wild type (WT) littermates. Contrary to our expectation, the BAPN-induced TAAD was severer in NgfSema3a/Sema3a mice than in wild-type (WT) mice. In addition, NgfSema3a/Sema3a mice showed higher aortic sympathetic innervation, inflammation and extracellular matrix degradation than the WT mice after BAPN treatment. The aortic vascular smooth muscle cells isolated from NgfSema3a/Sema3a mice and pretreated with BAPN in vivo for two weeks showed stronger capabilities of proliferation and migration than that from the WT mice. We conclude that the strategy of Ngf -driven Sema3a expression cannot suppress but worsens the BAPN-induced TAAD. By investigating the aortic phenotype of NgfSema3a/Sema3a mouse strain, we unexpectedly find a path to exacerbate BAPN-induced TAAD which might be useful in future TAAD studies.

Activation of the alternative complement pathway modulates inflammation in thoracic aortic aneurysm/dissection.

Thoracic aortic aneurysm/dissection (TAAD) is a lethal vascular disease, and several pathological factors participate in aortic medial degeneration. We previously discovered that the complement C3a-C3aR axis in smooth muscle cells promotes the development of thoracic aortic dissection (TAD) through regulation of matrix metalloproteinase 2. However, discerning the specific complement pathway that is activated and elucidating how inflammation of the aortic wall is initiated remain unknown. We ascertained that the plasma levels of C3a and C5a were significantly elevated in patients with TAD and that the levels of C3a, C4a, and C5a were higher in acute TAD than in chronic TAD. We also confirmed the activation of the complement in a TAD mouse model. Subsequently, knocking out Cfb (Cfb) or C4 in mice with TAD revealed that the alternative pathway and Cfb played a significant role in the TAD process. Activation of the alternative pathway led to generation of the anaphylatoxins C3a and C5a, and knocking out their receptors reduced the recruitment of inflammatory cells to the aortic wall. Moreover, we used serum from wild-type mice or recombinant mice Cfb as an exogenous source of Cfb to treat Cfb KO mice and observed that it exacerbated the onset and rupture of TAD. Finally, we knocked out Cfb in the FBN1C1041G/+ Marfan-syndrome mice and showed that the occurrence of TAA was reduced. In summary, the alternative complement pathway promoted the development of TAAD by recruiting infiltrating inflammatory cells. Targeting the alternative pathway may thus constitute a strategy for preventing the development of TAAD.NEW & NOTEWORTHY The alternative complement pathway promoted the development of TAAD by recruiting infiltrating inflammatory cells. Targeting the alternative pathway may thus constitute a strategy for preventing the development of TAAD.

Tomo-seq identifies NINJ1 as a potential target for anti-inflammatory strategy in thoracic aortic dissection.

BACKGROUND: Thoracic aortic dissection (TAD) is a life-threatening disease caused by an intimal tear in the aorta. The histological characteristics differ significantly between the tear area (TA) and the distant area. Previous studies have emphasized that certain specific genes tend to cluster at the TA. Obtaining a thorough understanding of the precise molecular signatures near the TA will assist in discovering therapeutic strategies for TAD. METHODS: We performed a paired comparison of the pathological patterns in the TA with that in the remote area (RA). We used Tomo-seq, genome-wide transcriptional profiling with spatial resolution, to obtain gene expression signatures spanning from the TA to the RA. Samples from multiple sporadic TAD patients and animal models were used to validate our findings. RESULTS: Pathological examination revealed that the TA of TAD exhibited more pronounced intimal hyperplasia, media degeneration, and inflammatory infiltration compared to the RA. The TA also had more apoptotic cells and CD31+α-SMA+ cells. Tomo-seq revealed four distinct gene expression patterns from the TA to the RA, which were inflammation, collagen catabolism, extracellular matrix remodeling, and cell stress, respectively. The spatial distribution of genes allowed us to identify genes that were potentially relevant with TAD. NINJ1 encoded the protein-mediated cytoplasmic membrane rupture, regulated tissue remodeling, showed high expression levels in the tear area, and co-expressed within the inflammatory pattern. The use of short hairpin RNA to reduce NINJ1 expression in the beta-aminopropionitrile-induced TAD model led to a significant decrease in TAD formation. Additionally, it resulted in reduced infiltration of inflammatory cells and a decrease in the number of CD31+α-SMA+ cells. The NINJ1-neutralizing antibody also demonstrated comparable therapeutic effects and can effectively impede the formation of TAD. CONCLUSIONS: Our study showed that Tomo-seq had the advantage of obtaining spatial expression information of TAD across the TA and the RA. We pointed out that NINJ1 may be involved in inflammation and tissue remodeling, which played an important role in the formation of TAD. NINJ1 may serve as a potential therapeutic target for TAD.

Multi-Omics of Familial Thoracic Aortic Aneurysm and Dissection: Calcium Transport Impairment Predisposes Aortas to Dissection.

Several genetic defects, including a mutation in myosin heavy chain 11 (Myh11), are reported to cause familial thoracic aortic aneurysm and dissection (FTAAD). We recently showed that mice lacking K1256 of Myh11 developed aortic dissection when stimulated with angiotensin II, despite the absence of major pathological phenotypic abnormalities prior to stimulation. In this study, we used a comprehensive, data-driven, unbiased, multi-omics approach to find underlying changes in transcription and metabolism that predispose the aorta to dissection in mice harboring the Myh11 K1256del mutation. Pathway analysis of transcriptomes showed that genes involved in membrane transport were downregulated in homozygous mutant (Myh11ΔK/ΔK) aortas. Furthermore, expanding the analysis with metabolomics showed that two mechanisms that raise the cytosolic Ca2+ concentration-multiple calcium channel expression and ADP-ribose synthesis-were attenuated in Myh11ΔK/ΔK aortas. We suggest that the impairment of the Ca2+ influx attenuates aortic contraction and that suboptimal contraction predisposes the aorta to dissection.

Single-Molecule Spatial Transcriptomics of Human Thoracic Aortic Aneurysms Uncovers Calcification-Related CARTPT-Expressing Smooth Muscle Cells.

BACKGROUND: Although single-cell RNA-sequencing is commonly applied to dissect the heterogeneity in human tissues, it involves the preparation of single-cell suspensions via cell dissociation, causing loss of spatial information. In this study, we employed high-resolution single-cell transcriptome imaging to reveal rare smooth muscle cell (SMC) types in human thoracic aortic aneurysm (TAA) tissue samples. METHODS: Single-molecule spatial distribution of transcripts from 140 genes was analyzed in fresh-frozen human TAA samples with region and sex-matched controls. In vitro studies and tissue staining were performed to examine human CART prepropeptide (CARTPT) regulation and function. RESULTS: We captured thousands of cells per sample including a spatially distinct CARTPT-expressing SMC subtype enriched in male TAA samples. Immunoassays confirmed human CART (cocaine- and amphetamine-regulated transcript) protein enrichment in male TAA tissue and truncated CARTPT secretion into cell culture medium. Oxidized low-density lipoprotein, a cardiovascular risk factor, induced CARTPT expression, whereas CARTPT overexpression in human aortic SMCs increased the expression of key osteochondrogenic transcription factors and reduced contractile gene expression. Recombinant human CART treatment of human SMCs further confirmed this phenotype. Alizarin red staining revealed calcium deposition in male TAA samples showing similar localization with human CART staining. CONCLUSIONS: Here, we demonstrate the feasibility of single-molecule imaging in uncovering rare SMC subtypes in the diseased human aorta, a difficult tissue to dissociate. We identified a spatially distinct CARTPT-expressing SMC subtype enriched in male human TAA samples. Our functional studies suggest that human CART promotes osteochondrogenic switch of aortic SMCs, potentially leading to medial calcification of the thoracic aorta.

Causal Role for Neutrophil Elastase in Thoracic Aortic Dissection in Mice.

BACKGROUND: Thoracic aortic dissection (TAD) is a life-threatening aortic disease without effective medical treatment. Increasing evidence has suggested a role for NE (neutrophil elastase) in vascular diseases. In this study, we aimed at investigating a causal role for NE in TAD and exploring the molecular mechanisms involved. METHODS: ß-aminopropionitrile monofumarate was administrated in mice to induce TAD. NE deficiency mice, pharmacological inhibitor GW311616A, and adeno-associated virus-2-mediated in vivo gene transfer were applied to explore a causal role for NE and associated target gene in TAD formation. Multiple functional assays and biochemical analyses were conducted to unravel the underlying cellular and molecular mechanisms of NE in TAD. RESULTS: NE aortic gene expression and plasma activity was significantly increased during ß-aminopropionitrile monofumarate-induced TAD and in patients with acute TAD. NE deficiency prevents ß-aminopropionitrile monofumarate-induced TAD onset/development, and GW311616A administration ameliorated TAD formation/progression. Decreased levels of neutrophil extracellular traps, inflammatory cells, and MMP (matrix metalloproteinase)-2/9 were observed in NE-deficient mice. TBL1x (F-box-like/WD repeat-containing protein TBL1x) has been identified as a novel substrate and functional downstream target of NE in TAD. Loss-of-function studies revealed that NE mediated inflammatory cell transendothelial migration by modulating TBL1x-LTA4H (leukotriene A4 hydrolase) signaling and that NE regulated smooth muscle cell phenotype modulation under TAD pathological condition by regulating TBL1x-MECP2 (methyl CpG-binding protein 2) signal axis. Further mechanistic studies showed that TBL1x inhibition decreased the binding of TBL1x and HDAC3 (histone deacetylase 3) to MECP2 and LTA4H gene promoters, respectively. Finally, adeno-associated virus-2-mediated Tbl1x gene knockdown in aortic smooth muscle cells confirmed a regulatory role for TBL1x in NE-mediated TAD formation. CONCLUSIONS: We unravel a critical role of NE and its target TBL1x in regulating inflammatory cell migration and smooth muscle cell phenotype modulation in the context of TAD. Our findings suggest that the NE-TBL1x signal axis represents a valuable therapeutic for treating high-risk TAD patients.

Epigenetic Induction of Smooth Muscle Cell Phenotypic Alterations in Aortic Aneurysms and Dissections.

BACKGROUND: Smooth muscle cell (SMC) phenotypic switching has been increasingly detected in aortic aneurysm and dissection (AAD) tissues. However, the diverse SMC phenotypes in AAD tissues and the mechanisms driving SMC phenotypic alterations remain to be identified. METHODS: We examined the transcriptomic and epigenomic dynamics of aortic SMC phenotypic changes in mice with angiotensin II-induced AAD by using single-cell RNA sequencing and single-cell sequencing assay for transposase-accessible chromatin. SMC phenotypic alteration in aortas from patients with ascending thoracic AAD was examined by using single-cell RNA sequencing analysis. RESULTS: Single-cell RNA sequencing analysis revealed that aortic stress induced the transition of SMCs from a primary contractile phenotype to proliferative, extracellular matrix-producing, and inflammatory phenotypes. Lineage tracing showed the complete transformation of SMCs to fibroblasts and macrophages. Single-cell sequencing assay for transposase-accessible chromatin analysis indicated that these phenotypic alterations were controlled by chromatin remodeling marked by the reduced chromatin accessibility of contractile genes and the induced chromatin accessibility of genes involved in proliferation, extracellular matrix, and inflammation. IRF3 (interferon regulatory factor 3), a proinflammatory transcription factor activated by cytosolic DNA, was identified as a key driver of the transition of aortic SMCs from a contractile phenotype to an inflammatory phenotype. In cultured SMCs, cytosolic DNA signaled through its sensor STING (stimulator of interferon genes)-TBK1 (tank-binding kinase 1) to activate IRF3, which bound and recruited EZH2 (enhancer of zeste homolog 2) to contractile genes to induce repressive H3K27me3 modification and gene suppression. In contrast, double-stranded DNA-STING-IRF3 signaling induced inflammatory gene expression in SMCs. In Sting-/- mice, the aortic stress-induced transition of SMCs into an inflammatory phenotype was prevented, and SMC populations were preserved. Finally, profound SMC phenotypic alterations toward diverse directions were detected in human ascending thoracic AAD tissues. CONCLUSIONS: Our study reveals the dynamic epigenetic induction of SMC phenotypic alterations in AAD. DNA damage and cytosolic leakage drive SMCs from a contractile phenotype to an inflammatory phenotype.

The Role of ADAMTS Proteoglycanases in Thoracic Aortic Disease.

Thoracic aortic aneurysm and dissection (TAAD) are complex disease states with high morbidity and mortality that pose significant challenges to early diagnosis. Patients with an aneurysm are asymptomatic and typically present to the emergency department only after the development of a dissection. The extracellular matrix (ECM) plays a crucial role in regulating the aortic structure and function. The histopathologic hallmark termed medial degeneration is characterised by smooth muscle cell (SMC) loss, the degradation of elastic and collagen fibres and proteoglycan (PG) accumulation. Covalently attached to the protein core of PGs are a number of glycosaminoglycan chains, negatively charged molecules that provide flexibility, compressibility, and viscoelasticity to the aorta. PG pooling in the media can produce discontinuities in the aortic wall leading to increased local stress. The accumulation of PGs is likely due to an imbalance between their synthesis by SMCs and decreased proteolysis by A Disintegrin-like and Metalloproteinase with Thrombospondin motifs (ADAMTS) proteoglycanases in the ECM. Mouse models of TAAD indicated that these proteases exert a crucial, albeit complex and not fully elucidated, role in this disease. This has led to a mounting interest in utilising ADAMTS proteoglycanases as biomarkers of TAAD. In this review, we discuss the role of ADAMTSs in thoracic aortic disease and their potential use in facilitating the clinical diagnosis of TAAD and disease progression.

SIRT6 is an epigenetic repressor of thoracic aortic aneurysms via inhibiting inflammation and senescence.

Thoracic aortic aneurysms (TAAs) develop asymptomatically and are characterized by dilatation of the aorta. This is considered a life-threating vascular disease due to the risk of aortic rupture and without effective treatments. The current understanding of the pathogenesis of TAA is still limited, especially for sporadic TAAs without known genetic mutation. Sirtuin 6 (SIRT6) expression was significantly decreased in the tunica media of sporadic human TAA tissues. Genetic knockout of Sirt6 in mouse vascular smooth muscle cells accelerated TAA formation and rupture, reduced survival, and increased vascular inflammation and senescence after angiotensin II infusion. Transcriptome analysis identified interleukin (IL)-1ß as a pivotal target of SIRT6, and increased IL-1ß levels correlated with vascular inflammation and senescence in human and mouse TAA samples. Chromatin immunoprecipitation revealed that SIRT6 bound to the Il1b promoter to repress expression partly by reducing the H3K9 and H3K56 acetylation. Genetic knockout of Il1b or pharmacological inhibition of IL-1ß signaling with the receptor antagonist anakinra rescued Sirt6 deficiency mediated aggravation of vascular inflammation, senescence, TAA formation and survival in mice. The findings reveal that SIRT6 protects against TAA by epigenetically inhibiting vascular inflammation and senescence, providing insight into potential epigenetic strategies for TAA treatment.

Senkyunolide I ameliorates thoracic aortic aneurysm and dissection in mice via inhibiting the oxidative stress and apoptosis of endothelial cells.

BACKGROUND: Thoracic aortic aneurysm and aortic dissection (TAAD) is one of the most fatal cardiovascular diseases. Senkyunolide I (SEI) is a component of traditional Chinese medicine with remarkable anti-inflammatory properties and exhibits remarkable protective effects, but its impact on TAAD remains unclear. Our study aimed to explore the role of SEI in a murine model of TAAD and further explore the immunopharmacological mechanism. METHODS AND MATERIALS: The in vivo model were assessed using echocardiography, gross anatomy, and tissue staining. Western blot and immunofluorescence were performed to evaluate the effects of SEI in vivo and in vitro. A SEI solution injection containing 1 % dimethyl sulfoxide (DMSO) was administered intraperitoneally to the TAAD model group, while a normal saline injection comprising 1 % DMSO was administered to the sham group. RESULTS: SEI prevented TAAD formation induced by BAPN/Ang II and reduced the TAAD incidence in mice. SEI treatment significantly inhibited the degradation of collagen and elastin fibers in the extracellular matrix. Furthermore, it reduced the expression of inflammatory factors in the aortic intima. Western blot analysis revealed that SEI-treated mice showed a significant decrease in apoptosis-related protein levels in the aorta compared with the TAAD group. PI3K, Akt, and mTOR in the SEI treatment group were significantly lower than in the model group. SEI could also attenuate H2O2-induced Human umbilical vein endothelial cells (HUVECs) damage and reverse the decline in migrant cells. The apoptosis of HUVECs was considerably reduced by the SEI treatment. CONCLUSIONS: Conclusively, SEI may alleviate the progression of TAAD by suppressing the PI3K/Akt/NF-κB signaling pathway. The SEI's ability to inhibit inflammation and oxidative stress opens the way to restore the function of endothelial cells and vascular homeostasis, and thus to provide novel and promising options for the treatment of TAAD patients.

Changes in transmural mass transport correlate with ascending thoracic aortic aneurysm diameter in a fibulin-4 E57K knockin mouse model.

Thoracic aortic aneurysm is characterized by dilation of the aortic diameter by greater than 50%, which can lead to dissection or rupture. Common histopathology includes extracellular matrix remodeling that may affect transmural mass transport, defined as the movement of fluids and solutes across the wall. We measured in vitro ascending thoracic aorta mass transport in a mouse model with partial aneurysm phenotype penetration due to a mutation in the extracellular matrix protein fibulin-4 [Fbln4E57K/E57K, referred to as MU-A (aneurysm) or MU-NA (no aneurysm)]. To push the aneurysm phenotype, we also included MU mice with reduced levels of lysyl oxidase [Fbln4E57K/E57K;Lox+/-, referred to as MU-XA (extreme aneurysm)] and compared all groups to wild-type (WT) littermates. The phenotype variation allows investigation of how aneurysm severity correlates with mass transport parameters and extracellular matrix organization. We found that MU-NA ascending thoracic aortae have similar hydraulic conductance (Lp) to WT, but 397% higher solute permeability (ω) for 4 kDa FITC-dextran. In contrast, MU-A and MU-XA ascending thoracic aortae have 44-68% lower Lp and similar ω to WT. The results suggest that ascending thoracic aortic aneurysm progression involves an initial increase in ω, followed by a decrease in Lp after the aneurysm has formed. All MU ascending thoracic aortae are longer and have increased elastic fiber fragmentation in the extracellular matrix. There is a negative correlation between diameter and Lp or ω in MU ascending thoracic aortae. Changes in mass transport due to elastic fiber fragmentation could contribute to aneurysm progression or be leveraged for treatment.NEW & NOTEWORTHY Transmural mass transport is quantified in the ascending thoracic aorta of mice with a mutation in fibulin-4 that is associated with thoracic aortic aneurysms. Fluid and solute transport depend on aneurysm severity, correlate with elastic fiber fragmentation, and may be affected by proteoglycan deposition. Transport properties of the ascending thoracic aorta are provided and can be used in computational models. The changes in mass transport may contribute to aneurysm progression or be leveraged for aneurysm treatment.

Smooth Muscle-Alpha Actin R149C Pathogenic Variant Downregulates Integrin Recruitment at Cell-Matrix Adhesions and Decreases Cellular Contractility.

Thoracic aortic aneurysm is found in patients with ACTA2 pathogenic variants. ACTA2 missense variants are associated with impaired aortic smooth muscle cell (SMC) contraction. This study tested the hypothesis that the Acta2R149C/+ variant alters actin isoform expression and decreases integrin recruitment, thus, reducing aortic contractility. Stress relaxation measurements in thoracic aortic rings showed two functional regimes with a reduction of stress relaxation in the aorta from Acta2R149C/+ mice at low tension, but not at high tension values. Contractile responses to phenylephrine and potassium chloride were 50% lower in Acta2R149C/+ mice than in wild-type (WT) mice. Additionally, SMC were immunofluorescently labeled for specific proteins and imaged by confocal or total internal reflection fluorescence microscopy. The quantification of protein fluorescence of Acta2R149C/+ SMC showed a downregulation in smooth muscle α-actin (SMα-actin) and a compensatory upregulation of smooth muscle γ-actin (SMγ-actin) compared to WT cells. These results suggest that downregulation of SMα-actin leads to reduced SMC contractility, while upregulation of SMγ-actin may lead to increased SMC stiffness. Decreased α5ß1 and α2ß1 integrin recruitment at cell-matrix adhesions further reduce the ability of mutant cells to participate in cell-matrix crosstalk. Collectively, the results suggest that mutant Acta2R149C/+ aortic SMC have reduced contractility and interaction with the matrix, which are potential long-term contributing factors to thoracic aortic aneurysms.

Analysis of immunogenic cell death in ascending thoracic aortic aneurysms based on single-cell sequencing data.

Background: At present, research on immunogenic cell death (ICD) is mainly associated with cancer therapy. Little is known about the role of ICD in cardiovascular disease, especially in ascending thoracic aortic aneurysms (ATAA). Method: ATAA single-cell RNA (scRNA) sequencing data were analyzed to identify the involved cell types and determine their transcriptomic characteristics. The chi-square test, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, Gene Set Enrichment Analysis (GSEA), and CellChat for cell-to-cell communication analysis from the Gene Expression Omnibus (GEO) database were used. Result: A total of 10 cell types were identified, namely, monocytes, macrophages, CD4 T/NK (CD4+ T cells and natural killer T cells), mast cells, B/Plasma B cells, fibroblasts, endothelial cells, cytotoxic T cells (CD8+ T cells, CTLs), vascular smooth muscle cells (vSMCs), and mature dendritic cells (mDCs). A large number of inflammation-related pathways were present in the GSEA results. A large number of ICD-related pathways were found in the KEGG enrichment analysis of differentially expressed genes in endothelial cells. The number of mDCs and CTLs in the ATAA group was significantly different from that in the control group. A total of 44 pathway networks were obtained, of which 9 were associated with ICD in endothelial cells (CCL, CXCL, ANNEXIN, CD40, IL1, IL6, TNF, IFN-II, GALECTIN). The most important ligand-receptor pair by which endothelial cells act on CD4 T/NK cells, CTLs and mDCs is CXCL12-CXCR4. The most important ligand-receptor pair by which endothelial cells act on monocytes and macrophages is ANXA1-FPR1. The most important ligand-receptor pair by which CD4 T/NK cells and CTLs act on endothelial cells is CCL5-ACKR1. The most important ligand-receptor pair that myeloid cells (macrophages, monocytes and mDCs) act on endothelial cells is CXCL8-ACKR1. Moreover, vSMCs and fibroblasts mainly promote inflammatory responses through the MIF signaling pathway. Conclusion: ICD is present in ATAA and plays an important role in the development of ATAA. The target cells of ICD may be mainly endothelial cells, in which the aortic endothelial cell ACKR1 receptor can not only promote T-cell infiltration through the CCL5 ligand but also promote myeloid cell infiltration through the CXCL8 ligand. ACKR1 and CXCL12 may become target genes for ATAA drug therapy in the future.

NOTCH1 Gene as a Novel Cause of Thoracic Aortic Aneurysm in Patients with Tricuspid Aortic Valve: Two Cases Reported.

Thoracic aortic aneurysms (TAA) consist of abnormal dilation or the widening of a portion of the ascending aorta, due to weakness or destructuring of the walls of the vessel and are potentially lethal. The congenital bicuspid aortic valve (BAV) is considered a risk factor for the development of TAA because asymmetric blood flow through the bicuspid aortic valve detrimentally influences the wall of the ascending aorta. NOTCH1 mutations have been associated with non-syndromic TAAs as a consequence of BAV, but little is known regarding its haploinsufficiency and its relationship with connective tissue abnormalities. We report two cases in which there is clear evidence that alterations in the NOTCH1 gene are the cause of TAA in the absence of BAV. On the one hand, we describe a 117 Kb deletion that includes a large part of the NOTCH1 gene and no other coding genes, suggesting that haploinsufficiency can be considered a pathogenic mechanism for this gene associated with TAA. In addition, we describe two brothers who carry two variants, one in the NOTCH1 gene and another in the MIB1 gene, corroborating the involvement of different genes of the Notch pathway in aortic pathology.

Low Zinc Alleviates the Progression of Thoracic Aortic Dissection by Inhibiting Inflammation.

Vascular inflammation triggers the development of thoracic aortic dissection (TAD). Zinc deficiency could dampen tissue inflammation. However, the role of zinc as a nutritional intervention in the progression of TAD remains elusive. In this study, we employed a classical ß-aminopropionitrile monofumarate (BAPN)-induced TAD model in mice treated with low zinc and observed that the TAD progression was greatly ameliorated under low zinc conditions. Our results showed that low zinc could significantly improve aortic dissection and rupture (BAPN + low zinc vs. BAPN, 36% vs. 100%) and reduce mortality (BAPN + low zinc vs. BAPN, 22% vs. 57%). Mechanically, low zinc attenuated the infiltration of macrophages and inhibited the expression of inflammatory cytokines, suppressed the phenotype switch of vascular smooth muscle cells from contractile to synthetic types, and eventually alleviated the development of TAD. In conclusion, this study suggested that low zinc may serve as a potential nutritional intervention approach for TAD prevention.

Cordycepin suppresses vascular inflammation, apoptosis and oxidative stress of arterial smooth muscle cell in thoracic aortic aneurysm with VEGF inhibition.

BACKGROUND: Thoracic aortic aneurysm (TAA) is a type of common and serious vascular disease, in which inflammation, apoptosis and oxidative stress are strongly involved in the progression. Cordycepin, a bioactive compound from Cordyceps militaris, exhibits anti-inflammatory and anti-oxidative activities. This study aimed to address the role and mechanism of cordycepin in TAA. METHODS: The thoracic aortas were perivascularly administrated with calcium chloride (CaCl2), and human aortic smooth muscle cells (HASMCs) were incubated with angiotensin II (Ang II) to simulate the TAA model in vivo and in vitro, respectively. The effect and mechanism of cordycepin in TAA were explored by hematoxylin and eosin (HE) staining, immunohistochemistry (IHC), immunofluorescence (IF), western blot, biochemical test, cell counting kit-8 (CCK-8), and terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) assays. RESULTS: Cordycepin improved the CaCl2-induced the aneurysmal alteration and disappearance of normal wavy elastic structures of the aorta tissues, TAA incidence and thoracic aortic diameter in rats, and Ang II-induced the cell viability of HASMCs. Cordycepin reversed the CaCl2-induced the relative protein expression of cleaved caspase 9, cleaved caspase 3, interleukin (IL)-6, tumor necrosis factor (TNF)-α and IL-1ß, and the relative levels of glutathione (GSH), malonaldehyde (MDA) and reactive oxygen species (ROS) in vivo, or Ang II-induced these changes in vitro. Mechanically, cordycepin reduced the relative protein expressions of vascular endothelial growth factor (VEGF), VEGF receptor 2 (VEGFR2), cluster of differentiation 31 (CD31) and endothelial nitric oxide synthase (eNOS) in the Ang II-induced HASMCs. Correspondingly, overexpression of VEGF increased the levels of the indicators involved in apoptosis, inflammation and oxidative stress, which were antagonized with the cordycepin incubation in the Ang II-induced HASMCs. CONCLUSION: Cordycepin inhibited apoptosis, inflammation and oxidative stress of TAA through the inhibition of VEGF.

ADAMTS-7 deficiency attenuates thoracic aortic aneurysm and dissection in mice.

Thoracic aortic aneurysm and dissection (TAAD) is a life-threatening cardiovascular disease with severe extracellular matrix (ECM) remodeling that lacks efficient early stage diagnosis and nonsurgical therapy. A disintegrin and metalloproteinase with thrombospondin motif 7 (ADAMTS-7) is recognized as a novel locus for human coronary artery atherosclerosis. Previous work by us and others showed that ADAMTS-7 promoted atherosclerosis, postinjury neointima formation, and vascular calcification. However, whether ADAMTS-7 is involved in TAAD pathogenesis is unknown. We aimed to explore the alterations in ADAMTS-7 expression in human and mouse TAAD, and investigate the role of ADAMTS-7 in TAAD formation. A case-control study of TAAD patients (N = 86) and healthy participants (N = 88) was performed. The plasma ADAMTS-7 levels were markedly increased in TAAD patients within 24 h and peaked in 7 days. A TAAD mouse model was induced with 0.5% ß-aminopropionitrile (BAPN) in drinking water. ELISA analysis of mouse plasma, Western blotting, and immunohistochemical staining of aorta showed an increase in ADAMTS-7 in the early stage of TAAD. Moreover, ADAMTS-7-deficient mice exhibited significantly attenuated TAAD formation and TAAD rupture-related mortality in both male and female mice, which was accompanied by reduced artery dilation and inhibited elastin degradation. ADAMTS-7 deficiency caused repressed inflammatory response and complement system activation during TAAD formation. An increase in plasma ADAMTS-7 is a novel biomarker for human TAAD. ADAMTS-7 deficiency attenuates BAPN-induced murine TAAD. ADAMTS-7 is a potential novel target for TAAD diagnosis and therapy. KEY MESSAGES: A case-control study revealed increased plasma ADAMTS-7 is a risk factor for TAAD. ADAMTS-7 was elevated in plasma and aorta at early stage of mouse TAAD. ADAMTS-7 knockout attenuated mouse TAAD formation and mortality in both sexes.