Heritable thoracic aortic disease: a literature review on genetic aortopathies and current surgical management.

Heritable thoracic aortic disease puts patients at risk for aortic aneurysms, rupture, and dissections. The diagnosis and management of this heterogenous patient population continues to evolve. Last year, the American Heart Association/American College of Cardiology Joint Committee published diagnosis and management guidelines for aortic disease, which included those with genetic aortopathies. Additionally, evolving research studying the implications of underlying genetic aberrations with new genetic testing continues to become available. In this review, we evaluate the current literature surrounding the diagnosis and management of heritable thoracic aortic disease, as well as novel therapeutic approaches and future directions of research.

Genetic liability to elevated circulating IP-10, IFNγ and SCGFß levels in relation to thoracic aortic aneurysm: A mendelian randomization study.

Inflammation is associated with thoracic aortic aneurysm (TAA) but the effects of each circulating inflammatory factor on TAA remain unclear. In this study, we explored the relationship between circulating inflammatory factors and TAA risk using Mendelian randomization (MR) approach based on summary statistics from the latest genome-wide association study (GWAS) of 41 circulating inflammatory factors in 8293 Finns and a GWAS involving 1351 TAA cases and 18,295 controls of European ancestry. In univariable MR, higher interferon gamma-induced protein 10 (IP-10) levels, higher interferon gamma (IFNγ) levels and higher stem cell growth factor beta (SCGFß) levels were associated with an increased risk of TAA (OR = 1.37, 95 % CI = 1.17-1.59, p = 7.42 × 10-5; OR = 1.43, 95 % CI = 1.19-1.74, p = 2.04 × 10-4; OR = 1.27, 95 % CI = 1.09-1.48, p = 2.40 × 10-3, respectively). In multivariable MR, the patterns of associations for the three cytokines remained adjusting for each other or smoking, but were attenuated differently with adjustment for other cardiovascular risk factors, especially for lipids and body mass index. Bidirectional MR approach did not identify any significant associations between cytokines and risk factors. Our results indicated that circulating cytokines may play mediation roles in the pathogenesis of TAA. Further studies are needed to determine whether these biomarkers can be used to prevent and treat TAA.

miRNA Regulation of Cell Phenotype and Parietal Remodeling in Atherosclerotic and Non-Atherosclerotic Aortic Aneurysms: Differences and Similarities.

Aortic aneurysms are a serious health concern as their rupture leads to high morbidity and mortality. Abdominal aortic aneurysms (AAAs) and thoracic aortic aneurysms (TAAs) exhibit differences and similarities in their pathophysiological and pathogenetic features. AAA is a multifactorial disease, mainly associated with atherosclerosis, characterized by a relevant inflammatory response and calcification. TAA is rarely associated with atherosclerosis and in some cases is associated with genetic mutations such as Marfan syndrome (MFS) and bicuspid aortic valve (BAV). MFS-related and non-genetic or sporadic TAA share aortic degeneration with endothelial-to-mesenchymal transition (End-Mt) and fibrosis, whereas in BAV TAA, aortic degeneration with calcification prevails. microRNA (miRNAs) contribute to the regulation of aneurysmatic aortic remodeling. miRNAs are a class of non-coding RNAs, which post-transcriptionally regulate gene expression. In this review, we report the involvement of deregulated miRNAs in the different aortic remodeling characterizing AAAs and TAAs. In AAA, miRNA deregulation appears to be involved in parietal inflammatory response, smooth muscle cell (SMC) apoptosis and aortic wall calcification. In sporadic and MFS-related TAA, miRNA deregulation promotes End-Mt, SMC myofibroblastic phenotypic switching and fibrosis with glycosaminoglycan accumulation. In BAV TAA, miRNA deregulation sustains aortic calcification. Those differences may support the development of more personalized therapeutic approaches.

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.

MFAP5 variant-induced multiple giant thoracic aortic aneurysm.

Heritable thoracic aortic aneurysms are complex conditions characterised by the dilation or rupture of the thoracic aorta, often occurring as an autosomal-dominant disorder associated with life-threatening complications. In this case report, we present a de novo variant, MFAP5 c.236_237insA (p.N79Kfs9), which is implicated in the development of inherited thoracic aortic aneurysm. The proband, a 15-year-old male, presented with recurrent cough, dull chest pain, chest distress, vomiting, and reduced activity tolerance, leading to the diagnosis of heritable thoracic aortic aneurysms. Whole-exome sequencing identified a novel heterozygous variant in MFAP5 (NM_003480, c.236_237insA, and p.N79Kfs9). MutationTester and PolyPhen-s predicted this variant to be damaging and disease-causing (probability = 1), while the SFIT score indicated protein damage (0.001). Structural analysis using the AlphaFold Protein structure database revealed that this mutation disrupted the N-linked glycosylation site, resulting in a frameshift, amino acid sequence alteration, and truncation of an essential protein site. To our knowledge, this is the first case report describing a young patient with heritable thoracic aortic aneurysm carrying the novel MFAP5 c.236_237insA (p.N79Kfs*9) variant. This variant represents the third identified mutation site associated with heritable thoracic aortic aneurysm. Given the high mortality and morbidity rates associated with thoracic aortic aneurysms, the prevention of severe and fatal complications is crucial in the clinical management of this condition. Our case highlights the importance of whole-exome sequencing and genetic screening in identifying potential pathogenic or likely pathogenic variants, particularly in early-onset patients with aortic dilation, to inform appropriate management strategies.

Outcomes After Extent I Thoracoabdominal Aortic Repair: Focus on Heritable Aortic Disease.

BACKGROUND: Crawford extent I thoracoabdominal aortic aneurysm (TAAA) repairs are increasingly performed by an endovascular approach, including in patients with heritable thoracic aortic disease (HTAD). We evaluated outcomes after open extent I TAAA repair in patients with and without HTAD. METHODS: This retrospective study included 992 patients (median age, 67 years; quartile 1-quartile 3, 57-73 years) who underwent extent I TAAA (1990-2022), stratified by the presence of HTAD (n = 177 [17.8%]). Patients with HTAD had genetic aortopathies or presented at age ≤50 years, and 35% (62 of 177) had Marfan syndrome. Logistic regression was used to identify predictors of operative death and adverse event, a composite of operative death and persistent (present at discharge) stroke, paraplegia, paraparesis, and renal failure necessitating dialysis. Long-term outcomes were analyzed with competing risks analysis. RESULTS: Patients with HTAD had lower rates of operative mortality (1.7% vs 7.0%, P = .01) and composite adverse event (2.8% vs 12.3%, P < .001) than non-HTAD patients. Most HTAD patients were discharged home (92.6% vs 76.9%, P < .001). Predictors of operative death were increasing age, aortic dissection, tobacco use, chronic symptoms, and rupture. Predictors for adverse event were increasing age, acute symptoms, chronic dissection, and rupture. Patients with HTAD had substantially better repair-failure-free survival (P < .001). CONCLUSIONS: Open extent I TAAA repair was effective in patients with HTAD, with low operative mortality and adverse event rates, better late survival, and excellent long-term durability, making a compelling argument for preferring open repair in these patients.

A highly penetrant ACTA2 mutation of thoracic aortic disease.

BACKGROUND: The role of ACTA2 mutations in Familial Aortic Disease has been increasingly recognized. We describe a highly penetrant variant (R118Q) in a family with aortic disease. CASE REPORT: A patient presented to us for elective repair of an ascending aortic aneurysm with a family history of his mother expiring after aortic dissection. Genetic testing revealed he was a heterozygous carrier of the ACTA2 missense mutation R118Q. Subsequently, all living family members were tested for this variant and a full medical history was obtained to compile a family tree for the variant and penetrance of an aortic event (defined as lifetime occurrence of aortic surgery / dissection). In total 9 family members were identified and underwent genetic testing with 7/9 showing presence of the ACTA2 R118Q mutation or an aortic event. All patients over the age of 50 (n = 4) had an aortic event. Those events occurred at ages 54, 55, 60, and 62 (mean event at 57.8 ± 3.9 years). Three family members with the variant under the age of 40 have not had an aortic event and most are undergoing regular aortic surveillance via CT scan. CONCLUSIONS: Existing studies of known ACTA2 mutations describe a 76% aortic event rate by 85 years old. The R118Q missense mutation is a less common ACTA2 variant, estimated to be found in about 5% of patients with known mutations. Prior studies have predicted the R118Q mutation to have a slightly decreased risk of aortic events compared to other ACTA2 mutations. In this family, however, we demonstrate 100% penetrance of aortic disease above age 50. In today's era of excellent outcomes in elective aortic surgery, our team aggressively offers elective repair. We advocate for strict aortic surveillance for patients with this variant and would consider elective aortic replacement at 4.5 cm, or at an even smaller diameter in patients with a strong family history of dissection who are identified with this mutation.

"Big Data" Analyses Underlie Clinical Discoveries at the Aortic Institute.

This issue of the Yale Journal of Biology and Medicine (YJBM) focuses on Big Data and precision analytics in medical research. At the Aortic Institute at Yale New Haven Hospital, the vast majority of our investigations have emanated from our large, prospective clinical database of patients with thoracic aortic aneurysm (TAA), supplemented by ultra-large genetic sequencing files. Among the fundamental clinical and scientific discoveries enabled by application of advanced statistical and artificial intelligence techniques on these clinical and genetic databases are the following: From analysis of Traditional "Big Data" (Large data sets). 1. Ascending aortic aneurysms should be resected at 5 cm to prevent dissection and rupture. 2. Indexing aortic size to height improves aortic risk prognostication. 3. Aortic root dilatation is more malignant than mid-ascending aortic dilatation. 4. Ascending aortic aneurysm patients with bicuspid aortic valves do not carry the poorer prognosis previously postulated. 5. The descending and thoracoabdominal aorta are capable of rupture without dissection. 6. Female patients with TAA do more poorly than male patients. 7. Ascending aortic length is even better than aortic diameter at predicting dissection. 8. A "silver lining" of TAA disease is the profound, lifelong protection from atherosclerosis. From Modern "Big Data" Machine Learning/Artificial Intelligence analysis: 1. Machine learning models for TAA: outperforming traditional anatomic criteria. 2. Genetic testing for TAA and dissection and discovery of novel causative genes. 3. Phenotypic genetic characterization by Artificial Intelligence. 4. Panel of RNAs "detects" TAA. Such findings, based on (a) long-standing application of advanced conventional statistical analysis to large clinical data sets, and (b) recent application of advanced machine learning/artificial intelligence to large genetic data sets at the Yale Aortic Institute have advanced the diagnosis and medical and surgical treatment of TAA.

Integrated bulk and scRNA sequence identified anoikis-related diagnostic biomarkers and potential association with immune infiltration in type A aortic dissection.

Type-A aortic dissection (TAAD) is common life-threatening cardiovascular diseases with high-morbidity and mortality but the concrete etiology of disease remains unclear, which might disturb or delay the early diagnosis for TAAD. Anoikis is a special form of programmed cell-death (PCD) induced by detachment of anchorage-dependent cells from the extracellular matrix (ECM) or neighboring cells, and has been widely applied to identify anoikis-related biomarkers for the prediction and prognosis in oncological fields. However, the specific roles of anoikis-related genes (ARGs) in TAAD remain unclear. In this study, we first identified and validated eight diagnostic ARGs for TAAD based on multiple RNA-sequence datasets, including CHEK2, HIF1A, HK2, HMGA1, SERPINA1, PTPN1, SLC2A1 and VEGFA. The comprehensive functional annotation was evaluated by the integrated functional enrichments analysis. We identified the activation of inflammatory-related pathways, metabolic reprogramming and angiogenesis, and the inhibition of cardiovascular development pathways in TAAD. Immune cell infiltration (ICI) analysis further demonstrated that innate immune-cells were more dominant than adaptive immune-cells in TAAD tissues, especially in macrophages, monocytes, activated-DC, NKT cells and CD56+dim NK cells. The cellular landscape was further validated by single-cell RNA sequence technology with significant associations with anoikis in TAAD patients. Four vital ARGs (HIF1A, HMGA1, SERPINA1 and VEGFA) were ultimately identified along with the changes of differentiation trajectory, and major expressions were conformably concentrated on Macro1-3, Mono1-2 and Mono4 subtypes. These findings provide a promising diagnostic biomarker for the accurately diagnosing the disease and would be helpful to further explore the potential pathogenesis with anoikis process for TAAD.

Transcriptomics Provides Novel Insights into the Regulatory Mechanism of IncRNA HIF1 A-AS1 on Vascular Smooth Muscle Cells.

INTRODUCTION: Thoracic aortic aneurysm is a potentially fatal disease with a strong genetic contribution. The dysfunction of vascular smooth muscle cells (VSMCs) contributes to the formation of this aneurysm. Although previous studies suggested that long non-coding ribonucleic acid (RNA) hypoxia inducible factor 1 α-antisense RNA 1 (HIF1A-AS1) exerted a vital role in the progression and pathogenesis of thoracic aortic aneurysm, we managed to find a new regulatory mechanism of HIF1A-AS1 in VSMCs via transcriptomics. METHODS: Cell viability was detected by the cell counting kit-8 assay. Cell apoptosis was assessed by Annexin V-fluorescein isothiocyanate/propidium iodide double staining. Transwell migration assay and wound healing assay were performed to check the migration ability of HIF1A-AS1 on VSMCs. The NextSeq XTen system (Illumina) was used to collect RNA sequencing data. Lastly, reverse transcription-quantitative polymerase chain reaction confirmed the veracity and reliability of RNA-sequencing results. RESULTS: We observed that overexpressing HIF1A-AS1 successfully promoted apoptosis, significantly altered cell cycle distribution, and greatly attenuated migration in VSMCs, further highlighting the robust promoting effects of HIF1A-AS1 to thoracic aortic aneurysm. Moreover, transcriptomics was implemented to uncover its underlying mechanism. A total of 175 differently expressed genes were identified, with some of them enriched in apoptosis, migration, and cell cycle-related pathways. Intriguingly, some differently expressed genes were noted in vascular development or coagulation function pathways. CONCLUSION: We suggest that HIF1A-AS1 mediated the progression of thoracic aortic aneurysm by not only regulating the function of VSMCs, but also altering vascular development or coagulation function.

Aortic Dissection and a Previously Unreported ACTA2 Missense Variant Mutation in a Young Patient: A Case Report.

Hereditary connective tissue disease is known to cause aortic lesions at an early age. Familial aortic aneurysm/dissection is caused due to an ACTA2 mutation that affects smooth muscle structure. We present a case of a 15-year-old boy with a mild developmental disorder in whom no abnormalities were identified on previous physical examinations. The patient presented with severe left heart failure, extensive dissection from the ascending aorta to the common iliac artery, and myocardial and cerebral infarctions. He underwent an urgent Bentall surgery. Six months later, the patient underwent surgical reconstruction of the abdominal aorta from the aortic arch and returned to normal daily activities. Pathological examination demonstrated the absence of elastic fibers but presence of abundant reticular fibers and mucopolysaccharides from the tunica intima to the media. Genetic testing revealed a heterozygous missense variant of the ACTA2 gene. To the best of our knowledge, this is the first sporadic case of structurally abnormal smooth muscle organization resulting in clinical symptoms with no previously reported pathogenicity.

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.

Redox Dysregulation of Vascular Smooth Muscle Sirtuin-1 in Thoracic Aortic Aneurysm in Marfan Syndrome.

BACKGROUND: Thoracic aortic aneurysms (TAAs) are abnormal aortic dilatations and a major cardiovascular complication of Marfan syndrome. We previously demonstrated a critical role for vascular smooth muscle (VSM) SirT1 (sirtuin-1), a lysine deacetylase, against maladaptive aortic remodeling associated with chronic oxidative stress and aberrant activation of MMPs (matrix metalloproteinases). METHODS: In this study, we investigated whether redox dysregulation of SirT1 contributed to the pathogenesis of TAA using fibrillin-1 hypomorphic mice (Fbn1mgR/mgR), an established model of Marfan syndrome prone to aortic dissection/rupture. RESULTS: Oxidative stress markers 3-nitrotyrosine and 4-hydroxynonenal were significantly elevated in aortas of patients with Marfan syndrome. Moreover, reversible oxidative post-translational modifications (rOPTM) of protein cysteines, particularly S-glutathionylation, were dramatically increased in aortas of Fbn1mgR/mgR mice, before induction of severe oxidative stress markers. Fbn1mgR/mgR aortas and VSM cells exhibited an increase in rOPTM of SirT1, coinciding with the upregulation of acetylated proteins, an index of decreased SirT1 activity, and increased MMP2/9 activity. Mechanistically, we demonstrated that TGFß (transforming growth factor beta), which was increased in Fbn1mgR/mgR aortas, stimulated rOPTM of SirT1, decreasing its deacetylase activity in VSM cells. VSM cell-specific deletion of SirT1 in Fbn1mgR/mgR mice (SMKO-Fbn1mgR/mgR) caused a dramatic increase in aortic MMP2 expression and worsened TAA progression, leading to aortic rupture in 50% of SMKO-Fbn1mgR/mgR mice, compared with 25% of Fbn1mgR/mgR mice. rOPTM of SirT1, rOPTM-mediated inhibition of SirT1 activity, and increased MMP2/9 activity were all exacerbated by the deletion of Glrx (glutaredoxin-1), a specific deglutathionylation enzyme, while being corrected by overexpression of Glrx or of an oxidation-resistant SirT1 mutant in VSM cells. CONCLUSIONS: Our novel findings strongly suggest a causal role of S-glutathionylation of SirT1 in the pathogenesis of TAA. Prevention or reversal of SirT1 rOPTM may be a novel therapeutic strategy to prevent TAA and TAA dissection/ruptures in individuals with Marfan syndrome, for which, thus far, no targeted therapy has been developed.

Circ_0008285 silencing suppresses angiotensin II-induced vascular smooth muscle cell apoptosis in thoracic aortic aneurysm via miR-150-5p/BASP1 axis.

BACKGROUND: Vascular smooth muscle cells (VSMCs) are the predominant cell type of the aortic middle layer, the abnormal number or function of which has been demonstrated to have a role in thoracic aortic aneurysm (TAA). Here, this study aimed to identify the function of circ_0008285 in VSMC apoptosis. METHODS: Human VSMCs were treated with angiotensin II (Ang II) for functional experiments. Cell counting kit-8, 5-ethynyl-2'-deoxyuridine (EdU), and flow cytometry were applied for function analysis. The interaction between miR-150-5p and circ_0008285 or brain acid-soluble protein 1 (BASP1) was also evaluated by dual-luciferase reporter assay and RNA immunoprecipitation assay. Exosomes were isolated by the commercial kit. RESULTS: A highly expressed circ_0008285 was observed in the aortic tissues of TAA patients and Ang-II-induced VSMCs. Circ_0008285 deficiency dramatically reversed Ang-II-induced proliferation arrest and apoptosis promotion in VSMCs. Circ_0008285 functionally targeted miR-150-5p. MiR-150-5p inhibition attenuated the inhibitory effects of circ_0008285 silencing on Ang-II-evoked apoptosis in VSMCs. BASP1 was verified to be a target of miR-150-5p, and was proved to attenuate miR-150-5p-triggered apoptosis arrest in Ang-II-stimulated VSMCs. Additionally, extracellular circ_0008285 was packaged into exosomes, which could be transferred into the recipient cells. CONCLUSION: Circ_0008285 silencing could suppress Ang-II-induced VSMCs apoptosis via miR-150-5p/BASP1 axis, adding further understanding of the pathogenesis of TAA.

Urate-Lowering Therapy Inhibits Thoracic Aortic Aneurysm and Dissection Formation in Mice.

BACKGROUND: Thoracic aortic aneurysm and dissection (TAAD) is a highly lethal vascular disease without effective drug therapy. Whether elevated serum concentrations of uric acid are involved in TAAD development remains unclear. METHODS: Serum uric acid levels were detected in different TAAD mouse models and patients. The urate-lowering drug allopurinol was administered in the drinking water of TAAD mice. Adenine diet-induced mice were established to investigate the role of hyperuricemia in TAAD formation and RNA-sequencing of thoracic aortas from these mice was performed. RESULTS: We found serum uric acid levels were elevated in various mouse TAAD models, including mice fed a ß-aminopropionitrile diet, Marfan mice with fibrillin-1 haploinsufficiency (Fbn1C1041G/+), and ApoE-/- mice infused with Ang II (angiotensin II), as well as in patients with TAAD. Administration of urate-lowering drug allopurinol in the drinking water significantly alleviated TAAD formation in ß-aminopropionitrile-treated mice, Fbn1C1041G/+ mice, and Ang II-infused ApoE-/- mice. Moreover, an adenine diet was used to induce hyperuricemia in mice. Intriguingly, a 4-week adenine diet feeding directly induced TAAD formation characterized by increased maximal thoracic aortic diameters and severe elastin degradation, which were ameliorated by allopurinol. Unbiased RNA-sequencing in mouse thoracic aortas suggested that FcγR (Fc gamma receptor) was upregulated upon adenine diet, but reciprocally repressed by allopurinol. Mechanistically, hyperuricemia activated FcγR-mediated ERK1/2 (extracellular signal-regulated kinase 1/2) phosphorylation to induce macrophage inflammation and TAAD development, which was abrogated by allopurinol or FcγR deficiency. CONCLUSIONS: This study uncovered an important and previously unrecognized role of hyperuricemia in mediating the pathogenesis of TAAD, and uric acid-lowering drug may represent a promising therapeutic approach for TAAD.

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.

Lineage-Specific Induced Pluripotent Stem Cell-Derived Smooth Muscle Cell Modeling Predicts Integrin Alpha-V Antagonism Reduces Aortic Root Aneurysm Formation in Marfan Syndrome Mice.

BACKGROUND: The role of increased smooth muscle cell (SMC) integrin αv signaling in Marfan syndrome (MFS) aortic aneurysm remains unclear. Herein, we examine the mechanism and potential efficacy of integrin αv blockade as a therapeutic strategy to reduce aneurysm progression in MFS. METHODS: Induced pluripotent stem cells (iPSCs) were differentiated into aortic SMCs of the second heart field (SHF) and neural crest (NC) lineages, enabling in vitro modeling of MFS thoracic aortic aneurysms. The pathological role of integrin αv during aneurysm formation was confirmed by blockade of integrin αv with GLPG0187 in Fbn1C1039G/+ MFS mice. RESULTS: iPSC-derived MFS SHF SMCs overexpress integrin αv relative to MFS NC and healthy control SHF cells. Furthermore, integrin αv downstream targets (FAK [focal adhesion kinase]/AktThr308/mTORC1 [mechanistic target of rapamycin complex 1]) were activated, especially in MFS SHF. Treatment of MFS SHF SMCs with GLPG0187 reduced p-FAK/p-AktThr308/mTORC1 activity back to control SHF levels. Functionally, MFS SHF SMCs had increased proliferation and migration compared to MFS NC SMCs and control SMCs, which normalized with GLPG0187 treatment. In the Fbn1C1039G/+ MFS mouse model, integrin αv, p-AktThr308, and downstream targets of mTORC1 proteins were elevated in the aortic root/ascending segment compared to littermate wild-type control. Mice treated with GLPG0187 (age 6-14 weeks) had reduced aneurysm growth, elastin fragmentation, and reduction of the FAK/AktThr308/mTORC1 pathway. GLPG0187 treatment reduced the amount and severity of SMC modulation assessed by single-cell RNA sequencing. CONCLUSIONS: The integrin αv-FAK-AktThr308 signaling pathway is activated in iPSC SMCs from MFS patients, specifically from the SHF lineage. Mechanistically, this signaling pathway promotes SMC proliferation and migration in vitro. As biological proof of concept, GLPG0187 treatment slowed aneurysm growth and p-AktThr308 signaling in Fbn1C1039G/+ mice. Integrin αv blockade via GLPG0187 may be a promising therapeutic approach to inhibit MFS aneurysmal growth.

Novel biomarkers associated with thoracic aortic disease.

BACKGROUND: Biomarkers might help to improve diagnosis, surveillance and risk stratification of thoracic aortic disease (TAD). We explored the association between a broad spectrum of cardiovascular biomarkers with clinical characteristics and thoracic aortic diameter in TAD patients. METHODS: Venous blood-samples were obtained in 158 clinically stable TAD patients visiting our outpatient clinic (2017-2020). TAD was defined as a thoracic aortic diameter ≥ 40 mm, or genetic confirmation (hereditary TAD). The cardiovascular panel III of the Olink multiplex platform was used for batch analysis of 92 proteins. A comparison was made between biomarker levels in patients with and without previous aortic dissection and/or surgery, and with and without hereditary TAD. Linear regression analyses were applied to identify (relative, normalized) biomarker concentrations associated with the absolute thoracic aortic diameter (ADmax), and thoracic aortic diameter indexed for body surface area (IDmax). RESULTS: Median age of study patients was 61.0 (IQR 50.3-68.8) years, 37.3% females. Mean ADmax and IDmax were 43.3 ± 5.4 mm and 21.3 ± 3.3 mm/m2. After multivariable adjustment, Matrix Metalloproteinase-3 (MMP-3) and Insulin-like growth factor binding protein 2 (IGFBP-2) showed a significant positive association with ADmax and IDmax, respectively. Patients with previous aortic surgery/dissection had higher N-terminal-pro hormone BNP (NTproBNP) (median 3.67 [IQR 3.01-3.99] vs 2.84 [2.32-3.26], p ≤0.001). Patients with hereditary TAD had higher Trem-like transcript protein 2 (TLT-2) (median 4.64 [IQR 4.45-4.84]) than those with non-heriditary TAD (4.40 [4.17-4.64]; p = 0.00042). CONCLUSIONS: Among a broad range of biomarkers, MMP-3 and IGFBP-2 were associated with disease severity in TAD patients. The pathophysiological pathways uncovered by these biomarkers, and their potential clinical use warrants further research.

Single-cell transcriptome analysis identifies Versican(+) myofibroblast as a hallmark for thoracic aortic aneurysm marked by activation of PI3K-AKT signaling pathway.

BACKGROUND: Thoracic aortic aneurysm (TAA) is a silent but dangerous cardiovascular disease. Understanding molecular mechanisms of TAA on single-cell level might provide new strategies for preventing and treating TAA. METHODS: Single-cell RNA sequencing was performed on control and aneurysmal thoracic aorta to find out specific cell clusters and cell types. Western blot and histological staining were used to verify the findings of single-cell transcriptome analysis. Characteristics of Versican (VCAN) overexpressed myofibroblast was evaluated through bioinformatic methods and experimental validation. RESULTS: A total of 3 control and 8 TAA specimens were used for single-cell transcriptome analysis including 48,128 thoracic aortic cells. Among these cells, we found out a specific cell cluster containing both hallmarks of smooth muscle cell (SMC) and fibroblast. Thus, we defined these cells as myofibroblast. Further single-cell transcriptome analysis identified VCAN as a cellular marker of myofibroblast. Western blot and histological staining revealed that VCAN(+) myofibroblast was significantly increased in TAA specimens compared with control individuals. Differential analysis, functional, pathway enrichment analysis and cell-cell communication analysis demonstrated that VCAN(+) myofibroblast was closely associated with previous reported TAA associated pathological process including SMC proliferation, SMC migration and extracellular matrix (ECM) disruption. Pathway analysis found out significant activation of PI3K-AKT signaling pathway within VCAN(+) myofibroblast, which was further confirmed by experimental validation. CONCLUSIONS: Single-cell RNA sequencing identified VCAN(+) myofibroblast as a typical cellular hallmark of TAA. These cells might participate in the pathogenesis of TAA through activation of PI3K-AKT signaling pathway to link SMC proliferation, SMC migration and ECM disruption.

Molecular Mechanisms in Genetic Aortopathy-Signaling Pathways and Potential Interventions.

Thoracic aortic disease affects people of all ages and the majority of those aged <60 years have an underlying genetic cause. There is presently no effective medical therapy for thoracic aneurysm and surgery remains the principal intervention. Unlike abdominal aortic aneurysm, for which the inflammatory/atherosclerotic pathogenesis is well established, the mechanism of thoracic aneurysm is less understood. This paper examines the key cell signaling systems responsible for the growth and development of the aorta, homeostasis of endothelial and vascular smooth muscle cells and interactions between pathways. The evidence supporting a role for individual signaling pathways in pathogenesis of thoracic aortic aneurysm is examined and potential novel therapeutic approaches are reviewed. Several key signaling pathways, notably TGF-ß, WNT, NOTCH, PI3K/AKT and ANGII contribute to growth, proliferation, cell phenotype and survival for both vascular smooth muscle and endothelial cells. There is crosstalk between pathways, and between vascular smooth muscle and endothelial cells, with both synergistic and antagonistic interactions. A common feature of the activation of each is response to injury or abnormal cell stress. Considerable experimental evidence supports a contribution of each of these pathways to aneurysm formation. Although human information is less, there is sufficient data to implicate each pathway in the pathogenesis of human thoracic aneurysm. As some pathways i.e., WNT and NOTCH, play key roles in tissue growth and organogenesis in early life, it is possible that dysregulation of these pathways results in an abnormal aortic architecture even in infancy, thereby setting the stage for aneurysm development in later life. Given the fine tuning of these signaling systems, functional polymorphisms in key signaling elements may set up a future risk of thoracic aneurysm. Multiple novel therapeutic agents have been developed, targeting cell signaling pathways, predominantly in cancer medicine. Future investigations addressing cell specific targeting, reduced toxicity and also less intense treatment effects may hold promise for effective new medical treatments of thoracic aortic aneurysm.