Somatic Variants Acquired Later in Life Associated with Thoracic Aortic Aneurysms: JAK2 V617F.

The JAK2 V617F somatic variant is a well-known driver of myeloproliferative neoplasms (MPN) associated with an increased risk for athero-thrombotic cardiovascular disease. Recent studies have demonstrated its role in the development of thoracic aortic aneurysm (TAA). However, limited clinical information and level of JAK2 V617F burden have been provided for a comprehensive evaluation of potential confounders. A retrospective genotype-first study was conducted to identify carriers of the JAK2 V617F variant from an internal exome sequencing database in Yale DNA Diagnostics Lab. Additionally, the overall incidence of somatic variants in the JAK2 gene across various tissue types in the healthy population was carried out based on reanalysis of SomaMutDB and data from the UK Biobank (UKBB) cohort to compare our dataset to the population prevalence of the variant. In our database of 12,439 exomes, 594 (4.8%) were found to have a thoracic aortic aneurysm (TAA), and 12 (0.049%) were found to have a JAK2 V617F variant. Among the 12 JAK2 V617F variant carriers, five had a TAA (42%), among whom four had an ascending TAA and one had a descending TAA, with a variant allele fraction ranging from 11.2% to 20%. Among these five patients, 60% were female, and average age at diagnosis was 70 (49-79). The mean ascending aneurysm size was 5.05 cm (range 4.6-5.5 cm), and four patients had undergone surgical aortic replacement or repair. UKBB data revealed a positive correlation between the JAK2 V617F somatic variant and aortic valve disease (effect size 0.0086, p = 0.85) and TAA (effect size = 0.004, p = 0.92), although not statistically significant. An unexpectedly high prevalence of TAA in our dataset (5/594, 0.84%) is greater than the prevalence reported before for the general population, supporting its association with TAA. JAK2 V617F may contribute a meaningful proportion of otherwise unexplained aneurysm patients. Additionally, it may imply a potential JAK2-specific disease mechanism in the developmental of TAA, which suggests a possible target of therapy that warrants further investigation.

Elucidating thoracic aortic dissection pathogenesis: The interplay of m1A-related gene expressions and miR-16-5p/YTHDC1 Axis in NLRP3-dependent pyroptosis.

The underlying molecular mechanisms of thoracic aortic dissection (TAD) remain incompletely understood. Recent insights into RNA methylation and microRNA-mediated gene regulation offer new avenues for exploring how these processes contribute to the pathophysiology of TAD, particularly through the modulation of pyroptosis and smooth muscle cell viability. This research aimed to elucidate the interplay of m1A-related gene expressions and miR-16-5p/YTHDC1 Axis in NLRP3-dependent pyroptosis, a mechanism implicated in the pathogenesis of TAD. We collected tissue samples from 28 human TAD patients and 8 healthy aortic group, as well as utilized a mouse model to replicate the disease. A combination of computational, in vitro, and in vivo methods was applied, including CIBERSORTx analysis, Pearson correlation, gene transfection using antagomiR-16-5p, siRNA, and several staining as well as cell culture techniques. Our analysis indicated two differentially expressed genes, ALKBH2 and YTHDC1. We found significant upregulation of has-miR-16-5p and downregulation of YTHDC1 at mRNA level in AD samples. Immune cell infiltration in TAD samples was examined using the CIBERSORTx database. We confirmed that YTHDC1 was a target of miR-16-5p, as evidenced by an inhibitory effect on luciferase activity. Inhibition of miR-16-5p enhanced SMC proliferation and promoted cell viability whilst downregulating NLRP3-pyroptosis. YTHDC1 expression was increased, and NLRP3-pyroptosis expressions were inhibited, suggesting miR-16-5p/YTHDC1 axis may involve the NLRP3-pyroptosis of the SMC. In vivo analyses confirmed the prevention of NLRP3-pyroptosis in middle layer of the thoracic aorta, implying that the miR-16-5p/YTHDC1 axis regulated SMC proliferation via NLRP3-pyroptosis signaling. Our findings underscored the anti-pyroptotic role of miR-16-5p/YTHDC1 axis in the pathogenesis of TAD, suggesting a potential therapeutic strategy via targeting YTHDC1 and suppressing miR-16-5p to inhibit NLRP3-dependent pyroptosis. Although further investigation is needed, these results relating to SMC proliferation are a significant step forward in understanding TAD.

Single-cell sequencing reveals the impact of endothelial cell PIEZO1 expression on thoracic aortic aneurysm.

INTRODUCTION: Thoracic aortic aneurysm (TAA) is a severe vascular disease that threatens human life, characterized by focal dilatation of the entire aortic wall, with a diameter 1.5 times larger than normal. PIEZO1, a mechanosensitive cationic channel, monitors mechanical stimulations in the environment, transduces mechanical signals into electrical signals, and converts them into biological signals to activate intracellular signaling pathways. However, the role of PIEZO1 in TAA is still unclear. METHODS: We analyzed a single-cell database to investigate the expression level of PIEZO1 in TAA. We constructed a conditional knockout mouse model of Piezo1 and used the PIEZO1 agonist Yoda1 to intervene in the TAA model mice established by co-administration of BAPN and ANG-II. Finally, we explored the effect of Yoda1 on TAA in vitro. RESULTS AND DISCUSSION: We observed decreased PIEZO1 expression in TAA at both RNA and protein levels. Single-cell sequencing identified a specific reduction in Piezo1 expression in endothelial cells. Administration of PIEZO1 agonist Yoda1 prevented the formation of TAA. In PIEZO1 endothelial cell conditional knockout mice, Yoda1 inhibited TAA formation by interfering with PIEZO1. In vivo and in vitro experiments demonstrated that the effect of Yoda1 on endothelial cells involved macrophage infiltration, extracellular matrix degradation, and neovascularization. This study highlights the role of PIEZO1 in TAA and its potential as a therapeutic target, providing opportunities for clinical translation.

Divergent and Compensatory Effects of BMP2 and BMP4 on the VSMC Phenotype and BMP4's Role in Thoracic Aortic Aneurysm Development.

Vascular smooth muscle cells (VSMCs) play a key role in aortic aneurysm formation. Bone morphogenetic proteins (BMPs) have been implicated as important regulators of VSMC phenotype, and dysregulation of the BMP pathway has been shown to be associated with vascular diseases. The aim of this study was to investigate for the first time the effects of BMP-4 on the VSMC phenotype and to understand its role in the development of thoracic aortic aneurysms (TAAs). Using the angiotensin II (AngII) osmotic pump model in mice, aortas from mice with VSMC-specific BMP-4 deficiency showed changes similar to AngII-infused aortas, characterised by a loss of contractile markers, increased fibrosis, and activation of matrix metalloproteinase 9. When BMP-4 deficiency was combined with AngII infusion, there was a significantly higher rate of apoptosis and aortic dilatation. In vitro, VSMCs with mRNA silencing of BMP-4 displayed a dedifferentiated phenotype with activated canonical BMP signalling. In contrast, BMP-2-deficient VSMCs exhibited the opposite phenotype. The compensatory regulation between BMP-2 and BMP-4, with BMP-4 promoting the contractile phenotype, appeared to be independent of the canonical signalling pathway. Taken together, these results demonstrate the impact of VSMC-specific BMP-4 deficiency on TAA development.

Bioengineered vascular grafts with a pathogenic TGFBR1 variant model aneurysm formation in vivo and reveal underlying collagen defects.

Thoracic aortic aneurysm (TAA) is a life-threatening vascular disease frequently associated with underlying genetic causes. An inadequate understanding of human TAA pathogenesis highlights the need for better disease models. Here, we established a functional human TAA model in an animal host by combining human induced pluripotent stem cells (hiPSCs), bioengineered vascular grafts (BVGs), and gene editing. We generated BVGs from isogenic control hiPSC-derived vascular smooth muscle cells (SMCs) and mutant SMCs gene-edited to carry a Loeys-Dietz syndrome (LDS)-associated pathogenic variant (TGFBR1A230T). We also generated hiPSC-derived BVGs using cells from a patient with LDS (PatientA230T/+) and using genetically corrected cells (Patient+/+). Control and experimental BVGs were then implanted into the common carotid arteries of nude rats. The TGFBR1A230T variant led to impaired mechanical properties of BVGs, resulting in lower burst pressure and suture retention strength. BVGs carrying the variant dilated over time in vivo, resembling human TAA formation. Spatial transcriptomics profiling revealed defective expression of extracellular matrix (ECM) formation genes in PatientA230T/+ BVGs compared with Patient+/+ BVGs. Histological analysis and protein assays validated quantitative and qualitative ECM defects in PatientA230T/+ BVGs and patient tissue, including decreased collagen hydroxylation. SMC organization was also impaired in PatientA230T/+ BVGs as confirmed by vascular contraction testing. Silencing of collagen-modifying enzymes with small interfering RNAs reduced collagen proline hydroxylation in SMC-derived tissue constructs. These studies demonstrated the utility of BVGs to model human TAA formation in an animal host and highlighted the role of reduced collagen modifying enzyme activity in human TAA formation.

Unveiling cellular and molecular aspects of ascending thoracic aortic aneurysms and dissections.

Ascending thoracic aortic aneurysm (ATAA) remains a significant medical concern, with its asymptomatic nature posing diagnostic and monitoring challenges, thereby increasing the risk of aortic wall dissection and rupture. Current management of aortic repair relies on an aortic diameter threshold. However, this approach underestimates the complexity of aortic wall disease due to important knowledge gaps in understanding its underlying pathologic mechanisms.Since traditional risk factors cannot explain the initiation and progression of ATAA leading to dissection, local vascular factors such as extracellular matrix (ECM) and vascular smooth muscle cells (VSMCs) might harbor targets for early diagnosis and intervention. Derived from diverse embryonic lineages, VSMCs exhibit varied responses to genetic abnormalities that regulate their contractility. The transition of VSMCs into different phenotypes is an adaptive response to stress stimuli such as hemodynamic changes resulting from cardiovascular disease, aging, lifestyle, and genetic predisposition. Upon longer exposure to stress stimuli, VSMC phenotypic switching can instigate pathologic remodeling that contributes to the pathogenesis of ATAA.This review aims to illuminate the current understanding of cellular and molecular characteristics associated with ATAA and dissection, emphasizing the need for a more nuanced comprehension of the impaired ECM-VSMC network.

KLF15 maintains contractile phenotype of vascular smooth muscle cells and prevents thoracic aortic dissection by interacting with MRTFB.

Thoracic aortic dissection (TAD) is a highly dangerous cardiovascular disorder caused by weakening of the aortic wall, resulting in a sudden tear of the internal face. Progressive loss of the contractile apparatus in vascular smooth muscle cells (VSMCs) is a major event in TAD. Exploring the endogenous regulators essential for the contractile phenotype of VSMCs may aid the development of strategies to prevent TAD. Krüppel-like factor 15 (KLF15) overexpression was reported to inhibit TAD formation; however, the mechanisms by which KLF15 prevents TAD formation and whether KLF15 regulates the contractile phenotype of VSMCs in TAD are not well understood. Therefore, we investigated these unknown aspects of KLF15 function. We found that KLF15 expression was reduced in human TAD samples and ß-aminopropionitrile monofumarate-induced TAD mouse model. Klf15KO mice are susceptible to both ß-aminopropionitrile monofumarate- and angiotensin II-induced TAD. KLF15 deficiency results in reduced VSMC contractility and exacerbated vascular inflammation and extracellular matrix degradation. Mechanistically, KLF15 interacts with myocardin-related transcription factor B (MRTFB), a potent serum response factor coactivator that drives contractile gene expression. KLF15 silencing represses the MRTFB-induced activation of contractile genes in VSMCs. Thus, KLF15 cooperates with MRTFB to promote the expression of contractile genes in VSMCs, and its dysfunction may exacerbate TAD. These findings indicate that KLF15 may be a novel therapeutic target for the treatment of TAD.

Region-specific delamination strength of ascending thoracic aortic aneurysm of elderly hypertensive patients with bicuspid and tricuspid aortic valves.

Both ageing and hypertension are clinical factors that may lead to a higher propensity for dissection or rupture of ascending thoracic aortic aneurysms (ATAAs). This study sought to investigate effect of valve morphology on regional delamination strength of ATAAs in the elderly hypertensive patients. Whole fresh ATAA samples were harvested from 23 hypertensive patients (age, 71 ± 8 years) who underwent elective aortic surgery. Peeling tests were performed to measure region-specific delamination strengths of the ATAAs, which were compared between patients with bicuspid aortic valve (BAV) and tricuspid aortic valve (TAV). The regional delamination strengths of the ATAAs were further correlated with patient ages and aortic diameters for BAV and TAV groups. In the anterior and right lateral regions, the longitudinal delamination strengths of the ATAAs were statistically significantly higher for BAV patients than TAV patients (33 ± 7 vs. 23 ± 8 mN/mm, p = 0.01; 30 ± 7 vs. 19 ± 9 mN/mm, p = 0.02). For both BAV and TAV patients, the left lateral region exhibited significantly higher delamination strengths in both directions than the right lateral region. Histology revealed that disruption of elastic fibers in the right lateral region of the ATAAs was more severe for the TAV patients than the BAV patients. A strong inverse correlation between longitudinal delamination strength and age was identified in the right lateral region of the ATAAs of the TAV patients. Results suggest that TAV-ATAAs are more vulnerable to aortic dissection than BAV-ATAAs for the elderly hypertensive patients. Regardless of valve morphotypes, the right lateral region may be a special quadrant which is more likely to initiate dissection when compared with other regions.

Analysis of Vascular Smooth Muscle Cells from Thoracic Aortic Aneurysms Reveals DNA Damage and Cell Cycle Arrest as Hallmarks in Bicuspid Aortic Valve Patients.

Thoracic aortic aneurysm (TAA) is mainly sporadic and with higher incidence in the presence of a bicuspid aortic valve (BAV) for unknown reasons. The lack of drug therapy to delay TAA progression lies in the limited knowledge of pathophysiology. We aimed to identify the molecular hallmarks that differentiate the aortic dilatation associated with BAV and tricuspid aortic valve (TAV). Aortic vascular smooth muscle cells (VSMCs) isolated from sporadic TAA patients with BAV or TAV were analyzed by mass spectrometry. DNA oxidative damage assay and cell cycle profiling were performed in three independent cohorts supporting proteomics data. The alteration of secreted proteins was confirmed in plasma. Stress phenotype, oxidative stress, and enhanced DNA damage response (increased S-phase arrest and apoptosis) were found in BAV-TAA patients. The increased levels of plasma C1QTNF5, LAMA2, THSB3, and FAP confirm the enhanced stress in BAV-TAA. Plasma FAP and BGN point to an increased inflammatory condition in TAV. The arterial wall of BAV patients shows a limited capacity to counteract drivers of sporadic TAA. The molecular pathways identified support the need of differential molecular diagnosis and therapeutic approaches for BAV and TAV patients, showing specific markers in plasma which may serve to monitor therapy efficacy.

A yeast based assay establishes the pathogenicity of novel missense ACTA2 variants associated with aortic aneurysms.

The ACTA2 gene codes for alpha-smooth muscle actin, a critical component of the contractile apparatus of the vascular smooth muscle cells. Autosomal dominant variants in the ACTA2 gene have been associated to familial non-syndromic thoracic aortic aneurysm/dissection (TAAD). They are thought to act through a dominant-negative mechanism. These variants display incomplete penetrance and variable expressivity, complicating the validation of ACTA2 variants pathogenicity by family segregation studies. In this study, we developed a yeast based assay to test putative TAAD-associated ACTA2 variants. We identified five new heterozygous ACTA2 missense variants in TAAD patients through next generation sequencing. We decided to test their pathogenicity in Saccharomyces cerevisiae, since yeast actin is very similar to human alpha-smooth muscle actin, and the residues at which the TAAD-associated variants occur in ACTA2 are well conserved. A wild type yeast strain was transformed with a vector expressing the different mutant alleles, to model the heterozygous condition of patients. Then, we evaluated yeast growth by spot test and cytoskeletal and mitochondrial morphology by fluorescence microscopy. We found that mutant yeast strains displayed only mild growth defects but a significant increase in the percentage of cells with abnormal mitochondrial distribution and abnormal organization of the actin cytoskeleton compared to controls. All variants appeared to interfere with the activity of wild type actin in yeast, suggesting a dominant-negative pathogenic mechanism. Our results demonstrate the utility of using the yeast actin model system to validate the pathogenicity of TAAD-associated ACTA2 variants.

Integrative multi-omics analyses reveal vesicle transport as a potential target for thoracic aortic aneurysm.

BACKGROUND: Thoracic aortic aneurysm (TAA) refers to dilation and enlargement of the thoracic aorta caused by various reasons. Most patients have no apparent symptoms in the early stage and are subject to a poor prognosis once the aneurysm ruptures. It is crucial to identify individuals who are predisposed to TAA and to discover effective therapeutic targets for early intervention. METHODS: We conducted a label-free quantitative proteomic analysis among aorta tissue samples from TAA patients to screen differentially expressed proteins (DEPs) and key co-expression modules. Two datasets from Gene Expression Omnibus (GEO) database were included for integrative analysis, and the identified genes were subjected to immunohistochemistry (IHC) validation. Detailed vesicle transport related enrichment analysis was conducted and two FDA-approved drugs, chlorpromazine (CPZ) and chloroquine (CQ), were selected for in vivo inhibition of vesicle transport in mice TAA model. The diameter of thoracic aorta, mortality and histological differences after interventions were evaluated. RESULTS: We found significant enrichments in functions involved with vesicle transport, extracellular matrix organizing, and infection diseases in TAA. Endocytosis was the most essential vesicle transport process in TAA formation. Interventions with CPZ and CQ significantly reduced the aneurysm diameter and elastin degradation in vivo and enhanced the survival rates of TAA mice. CONCLUSIONS: We systematically screened the aberrantly regulated bioprocesses in TAA based on integrative multi-omics analyses, identified and demonstrated the importance of vesicle transport in the TAA formation. Our study provided pilot evidence that vesicular transport was a potential and promising target for the treatment of TAA.

Are telocytes related to maintenance of vascular homeostasis in normal and pathological aorta?

The telocyte (TC) is a new interstitial cell type described in a wide variety of organs and loose connective tissues around small vessels, but its presence in large arteries remains unexplored. TCs have small cell bodies and remarkably thin, long, moniliform processes called telopods (Tps). Using transmission electron microscopy and immunofluorescence, we identified TCs in normal human thoracic aortas and in those with aneurysm or acute dissection (TAAD). In normal aortas the TCs were distributed throughout the connective tissue of the adventitial layer, in its innermost portion and at the zone of transition with the medial layer, with their long axes oriented parallel to the external elastic lamellae, forming a three-dimensional network, without prevalence in the media layer. In contrast, TAAD TCs were present in the medial layer and in regions of neovascularization. The most important feature of the adventitia of diseased aortas was the presence of numerous contacts between TCs and stem cells, including vascular progenitor cells. Although the biologically functional correlations need to be elucidated, the morphological observations presented here provide strong evidence of the involvement of TCs in maintaining vascular homeostasis in pathological situations of tissue injury.

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.

Morphological risk of acute type A aortic dissection in the mildly to moderately dilated aorta.

OBJECTIVES: This study aimed to analyse and determine the role of aortic length and curvature in the pathogenesis of acute type A aortic dissection (ATAAD) with ascending aortic diameters (AADs) <5 cm. METHODS: We reviewed the clinical and imaging data of patients with ATAAD (n = 201) and ascending aortic dilation (n = 83). Thoracic aortic bending index (TABI) was used to quantify aortic curvature and analyse its role in ATAAD below the diameter risk threshold. RESULTS: The AAD was <5.0 and <4.0 cm in 78% and 37% of patients with ATAAD, respectively. The median ascending aortic length (AAL) was 104.6 mm (Q1-Q3, 96.5-113.6 mm), and in 62.7% of patients, it was <11 cm. The median TABI was 14.99 mm/cm (Q1-Q3, 14.18-15.86 mm/cm). Patients with ATAAD and those with aortic dilation were matched for AAD, age, sex, height and other clinical factors. After matched, the dissection group had higher AALs (median, 102.9 mm; Q1-Q3, 96.0-112.5 mm vs median, 88.2 mm; Q1-Q3, 83.7-95.9 mm; P < 0.001) and TABI (median, 14.84 mm/cm; Q1-Q3, 14.06-15.83 mm/cm vs median, 13.55 mm/cm; Q1-Q3, 13.03-14.28 mm/cm; P < 0.001). According to the regression analysis, the area under the curve required to distinguish patients with ATAAD from those with aortic dilation was 0.831 in AAL, 0.837 in TABI and 0.907 when AAL was combined with TABI. CONCLUSIONS: The patients with ATAAD had higher AAL and TABI than those with aortic dilation. The combination of TABI and AAL might be a potential morphological marker for determining ATAAD risk below the current aortic diameter risk threshold.

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.

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.

Loss of TIMP3, but not TIMP4, exacerbates thoracic and abdominal aortic aneurysm.

AIMS: Aorta exhibits regional heterogeneity (structural and functional), while different etiologies for thoracic and abdominal aortic aneurysm (TAA, AAA) are recognized. Tissue inhibitor of metalloproteinases (TIMPs) regulate vascular remodeling through different mechanisms. Region-dependent functions have been reported for TIMP3 and TIMP4 in vascular pathologies. We investigated the region-specific function of these TIMPs in development of TAA versus AAA. METHODS & RESULTS: TAA or AAA was induced in male and female mice lacking TIMP3 (Timp3-/-), TIMP4 (Timp4-/-) or in wildtype (WT) mice by peri-adventitial elastase application. Loss of TIMP3 exacerbated TAA and AAA severity in males and females, with a greater increase in proteinase activity, smooth muscle cell phenotypic switching post-AAA and -TAA, while increased inflammation was detected in the media post-AAA, but in the adventitia post-TAA. Timp3-/- mice showed impaired intimal barrier integrity post-AAA, but a greater adventitial vasa-vasorum branching post-TAA, which could explain the site of inflammation in AAA versus TAA. Severity of TAA and AAA in Timp4-/- mice was similar to WT mice. In vitro, Timp3 knockdown more severely compromised the permeability of human aortic EC monolayer compared to Timp4 knockdown or the control group. In aneurysmal aorta specimens from patients, TIMP3 expression decreased in the media in AAA, and in adventitial in TAA specimens, consistent with the impact of its loss in AAA versus TAA in mice. CONCLUSION: TIMP3 loss exacerbates inflammation, adverse remodeling and aortic dilation, but triggers different patterns of remodeling in AAA versus TAA, and through different mechanisms.

Regional biomechanical characterization of human ascending aortic aneurysms: Microstructure and biaxial mechanical response.

The ascending thoracic aortic aneurysm (ATAA) is a permanent dilatation of the vessel with a high risk of adverse events, and shows heterogeneous properties. To investigate regional differences in the biomechanical properties of ATAAs, tissue samples were collected from 10 patients with tricuspid aortic valve phenotype and specimens from minor, anterior, major, and posterior regions were subjected to multi-ratio planar biaxial extension tests and second-harmonic generation (SHG) imaging. Using the data, parameters of a microstructure-motivated constitutive model were obtained considering fiber dispersion. SHG imaging showed disruptions in the organization of the layers. Structural and material parameters did not differ significantly between regions. The non-symmetric fiber dispersion model proposed by Holzapfel et al. [25] was used to fit the data. The mean angle of collagen fibers was negatively correlated between minor and anterior regions, and the parameter associated with collagen fiber stiffness was positively correlated between minor and major regions. Furthermore, correlations were found between the stiffness of the ground matrix and the mean fiber angle, and between the parameter associated with the collagen fiber stiffness and the out-of-plane dispersion parameter in the posterior and minor regions, respectively. The experimental data collected in this study contribute to the biomechanical data available in the literature on human ATAAs. Region-specific parameters for the constitutive models are fundamental to improve the current risk stratification strategies, which are mainly based on aortic size. Such investigations can facilitate the development of more advanced finite element models capable of capturing the regional heterogeneity of pathological tissues. STATEMENT OF SIGNIFICANCE: Tissue samples of human ascending thoracic aortic aneurysms (ATAA) were collected. Samples from four regions underwent multi-ratio planar biaxial extension tests and second-harmonic generation imaging. Region-specific parameters of a microstructure-motivated model considering fiber dispersion were obtained. Structural and material parameters did not differ significantly between regions, however, the mean fiber angle was negatively correlated between minor and anterior regions, and the parameter associated with collagen fiber stiffness was positively correlated between minor and major regions. Furthermore, correlations were found between the stiffness of the ground matrix and the mean fiber angle, and between the parameter associated with the collagen fiber stiffness and the out-of-plane dispersion parameter in the posterior and minor regions, respectively. This study provides a unique set of mechanical and structural data, supporting the microstructural influence on the tissue response. It may facilitate the development of better finite element models capable of capturing the regional tissue heterogeneity.

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.