Thoracic Aortic Aneurysm Tissue Research Articles

CircCDYL Contributes to Apoptosis, Ferroptosis, and Oxidative Stress of Ang II-Induced Vascular Smooth Muscle Cells in Thoracic Aortic Aneurysm.

Circular RNAs (circRNAs) have important regulation in thoracic aortic aneurysm (TAA). The function and mechanism of circCDYL (circ_0008285) was explored in TAA here. Angiotensin II (Ang II) was used to construct a TAA model. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed for the detection of circCDYL, miR-1270, and a disintegrin and metalloproteinase 10 (ADAM10). Cell viability was examined via cell counting kit-8 (CCK-8) assay and proliferation was analyzed using Ethynyl-2'-deoxyuridine (EdU) assay. Apoptosis rate was assessed via flow cytometry. Western blot was used for protein detection. Oxidative stress was evaluated by commercial kits. CircCDYL was upregulated in TAA tissues and Ang II-induced circCDYL upregulation in vascular smooth muscle cells (VSMCs). Knockdown of circCDYL weakened Ang II-aroused inhibition of viability, proliferation, and promotion of apoptosis, ferroptosis, and oxidative stress in VSMCs. CircCDYL served as a miR-1270 sponge. The mitigated regulation of circCDYL knockdown for Ang II-induced injury was restored after miR-1270 downregulation. CircCDYL positively regulated ADAM10 through interacting with miR-1270. Overexpression of miR-1270 abated Ang II-induced injury by downregulating ADAM10. In conclusion, circCDYL was involved in the Ang II-induced VSMC injury in TAA via the miR-1270/ADAM10 axis.

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

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. 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. 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. 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.

Thoracic Aortic Aneurysm Risk Assessment: A Machine Learning Approach

BackgroundTraditional methods of risk assessment for thoracic aortic aneurysm (TAA) based on aneurysm size alone have been called into question as being unreliable in predicting complications. Biomechanical function of aortic tissue may be a better predictor of risk, but it is difficult to determine in vivo. ObjectivesThis study investigates using a machine learning (ML) model as a correlative measure of energy loss, a measure of TAA biomechanical function. MethodsBiaxial tensile testing was performed on resected TAA tissue collected from patients undergoing surgery. The energy loss of the tissue was calculated and used as the representative output. Input parameters were collected from clinical assessments including observations from medical scans and genetic paneling. Four ML algorithms including Gaussian process regression were trained in Matlab. ResultsA total of 158 patients were considered (mean age 62 years, range 22-89 years, 78% male), including 11 healthy controls. The mean ascending aortic diameter was 47 ± 10 mm, with 46% having a bicuspid aortic valve. The best-performing model was found to give a greater correlative measure to energy loss (R2 = 0.63) than the surprisingly poor performance of aortic diameter (R2 = 0.26) and indexed aortic size (R2 = 0.32). An echocardiogram-derived stiffness metric was investigated on a smaller subcohort of 67 patients as an additional input, improving the correlative performance from R2 = 0.46 to R2 = 0.62. ConclusionsA preliminary set of models demonstrated the ability of a ML algorithm to improve prediction of the mechanical function of TAA tissue. This model can use clinical data to provide additional information for risk stratification.

Circ_0000595 knockdown alleviates CoCl2-mediated effects in VSMCs by regulating the miR-582-3p/ADAM10 axis.

Thoracic aortic aneurysm (TAA) is a serious vascular disease causing the death of elder people. Accumulating studies have reported that circular RNAs (circRNAs) are implicated in the regulation of aortic aneurysms. However, the role of circ_0000595 in the progression of TAA is still unclear. Quantitative real-time PCR (qRT-PCR) and western blotting were implemented to assess circ_0000595, microRNA (miR)-582-3p, guanine nucleotide-binding protein alpha subunit (ADAM10), PCNA, Bax, and Bcl-2 expression. The proliferation of vascular smooth muscle cells was determined using cell counting kit 8 (CCK-8) and 5-ethynyl-2-deoxyuridine (EdU). Cell apoptosis was measured using flow cytometry, and caspase-3 activity was analyzed using a commercial kit. After bioinformatics analysis, the interaction between miR-582-3p and circ_0000595 or ADAM10 was validated using a dual-luciferase reporter and RNA immunoprecipitation. As compared with controls, TAA tissues and CoCl2-induced VSMCs displayed high expression of circ_0000595 and ADAM10, and low expression of miR-582-3p. CoCl2 treatment evidently suppressed VSMC proliferation and promoted VSMCs apoptosis, and these impacts were reverted by circ_0000595 knockdown. Circ_0000595 acted as a molecular sponge for miR-582-3p, and circ_0000595 silencing-mediated influences in CoCl2-induced VSMCs were overturned by miR-582-3p inhibitor. ADAM10 was confirmed as a target gene of miR-582-3p, and miR-582-3p overexpression-induced influence was almost restored by overexpressed ADAM10 in CoCl2-induced VSMCs. Besides, circ_0000595 contributed to ADAM10 protein expression by sponging miR-582-3p. Our data verified that circ_0000595 silencing might attenuate CoCl2-mediated impacts in VSMCs by regulating the miR-582-3p/ADAM10 axis, providing new potential roads for treating TAA.

HOXA cluster antisense RNA 2 elevates KIAA1522 expression through microRNA-520d-3p and insulin like growth factor 2 mRNA binding protein 3 to promote the growth of vascular smooth muscle cells in thoracic aortic aneurysm.

Recently, long non-coding RNAs (lncRNAs) have been revealed to mediate smooth muscle dysfunction in thoracic aortic aneurysm (TAA). LncRNA HOXA-AS2 has been proposed to engage in the regulation of diverse diseases. However, its function in TAA remains unknown. This study aimed to reveal the role and mechanism of HOXA-AS2 in VSMCs which were implicated in TAA formation. RT-qPCR or western blot was performed to detect RNA or protein expression levels. The role of HOXA-AS2 in VSMCs was explored by functional assays. The relationship among HOXA-AS2/miR-520d-3p/KIAA1522/IGF2BP3 was analysed via mechanism assays. HOXA-AS2 was detected to have significantly high expression in TAA tissues and function as an oncogene to promote proliferation of VSMCs, while inhibiting cell apoptosis (Figure 1, **P<0.01). HOXA-AS2 was unveiled to bind with miR-520d-3p (Figure 2, *P<0.05, **P<0.01) and further up-regulate KIAA1522 to facilitate the growth of VSMCs (Figure 3-4, *P<0.05, **P<0.01). HOXA-AS2 was also found to recruit IGF2BP3 to stabilize KIAA1522 mRNA (Figure 5, **P<0.01). All data were displayed as mean±standard deviation. HOXA-AS2 up-regulates KIAA1522 through targeting miR-520d-3p/IGF2BP3 to drive VSMC growth in TAA.

Integrating Bulk Transcriptome and Single-Cell RNA Sequencing Data Reveals the Landscape of the Immune Microenvironment in Thoracic Aortic Aneurysms.

Thoracic aortic aneurysm (TAA) is a life-threatening cardiovascular disease whose formation is reported to be associated with massive vascular inflammatory responses. To elucidate the roles of immune cell infiltration in the pathogenesis underlying TAA, we utilized multiple TAA datasets (microarray data and scRNA-seq data) and various immune-related algorithms (ssGSEA, CIBERSORT, and Seurat) to reveal the landscapes of the immune microenvironment in TAA. The results exhibited a significant increase in the infiltration of macrophages and T cells, which were mainly responsible for TAA formation among the immune cells. To further reveal the roles of immunocytes in TAA, we inferred the intercellular communications among the identified cells of aortic tissues. Notably, we found that in both normal aortic tissue and TAA tissue, the cells that interact most frequently are macrophages, endothelial cells (ECs), fibroblasts, and vascular smooth muscle cells (VSMCs). Among the cells, macrophages were the most prominent signal senders and receivers in TAA and normal aortic tissue. These findings suggest that macrophages play an important role in both the physiological and pathological conditions of the aorta. The present study provides a comprehensive evaluation of the immune cell composition and reveals the intercellular communication among aortic cells in human TAA tissues. These findings improve our understanding of TAA formation and progression and facilitate the development of effective medications to treat these conditions.

Explicit consideration of fiber recruitment in vascular constitutive formulation using beta functions

Constitutive models are of fundamental importance for describing the mechanical behavior of vascular tissues. In this article, we present a constitutive model that explicitly accounts for collagen fiber recruitment. The formulation, developed based two experimental observations, consists in a systematic use of beta functions. The distribution of random waviness is modeled by beta distribution, and the fiber stresses are represented by incomplete beta functions which are shown to be flexible in capturing different types of responses data. A four-point dispersion model, derived in the context of planar von Mises distribution, is developed to take into account of the orientation distribution of fibers. The dispersion weights appear as Bernstein polynomials of a single dispersion parameter. The descriptive and predictive capabilities are assessed using three groups of experimental data encompassing the responses of porcine thoracic aorta, human intracranial aneurysm, and human ascending thoracic aortic aneurysm tissues. The descriptive performance is evaluated by the quality of constitutive fitting. The predictive ability is tested based on the accuracy in predicting unfitted responses as well as in finite element simulations. The assessment indicates that the model has excellent descriptive and predictive capabilities.

Assessment of Computational Histopathology in Thoracic Aortic Aneurysm

Background and Hypothesis: Thoracic aortic aneurysm (TAA) histopathology includes elastic fiber (EF) abnormalities, mucoid extracellular matrix (MECM) accumulation, and smooth muscle derangement in the aortic medial layer. While semi-quantitative grading of these characteristics is a standard practice, computational characterization of medial layer components may facilitate novel quantitative analyses at higher throughput. We hypothesized that computational results would correlate with results of semi-quantitative grading of aortic histopathology.&#x0D; Experimental Design: Formalin-fixed, paraffin-embedded human aortic tissue sections were stained with Movat’s pentachrome to characterize aortic microstructure. Sections were also immunostained for nitrotyrosine residues to assess oxidative stress. Samples were initially graded semi-quantitatively by two independent blinded readers. Next, computational histopathology software was used a) to quantify the proportions of EF, MECM, and cellular area in the medial layer of pentachrome-stained sections and b) to quantify the distribution and intensity of positive nitrotyrosine staining in immunostained sections. Association between semi-quantitative grading and computed values was tested with ANOVA.&#x0D; Results: The cohort included 74 participants who underwent prophylactic aortic replacement for TAA and 23 healthy controls. The mean age was 54±17 years. On average, EFs accounted for 49% (range 6-90%) of medial tissue area, whereas MECM accounted for 25% (1-73%). The overall semi-quantitative grade of medial degeneration severity was associated with decrease in EF fraction (p=0.02). The grade of EF thinning also strongly correlated with decrease in EF fraction (p=1x10-6). Meanwhile, grade for accumulation of MECM was associated with increase in MECM (p=0.004). Increased semi-quantitative grading for nitrotyrosine levels was associated with increased nuclear signal optical density (p=9x10-10) and greater percentage of cells labeled as strongly positive (p=8x10-10).&#x0D; Conclusion and Potential Impact: We observed significant correlations between computed quantitative values and semi-quantitative grading. This suggests that computational histopathology is a valid method for investigation of human TAA tissues.

NEAT1 Boosts the Development of Thoracic Aortic Aneurysm Through Targeting miR-324-5p/RAN

Long non-coding RNAs (lncRNAs) have been identified in multiple cancers. Recently, NEAT1 is found to be up regulated in cervical cancer. Since the relationship between NEAT1 and thoracic aortic aneurysm (TAA) has not been clarified, our study focused on the role of NEAT1 in TAA. Bioinformatics, RNA pulls down and luciferase reporter assay were used to discover and determine miR-324-5p to be a target of NEAT1. RT-qPCR was used to examine NEAT1, RAN and miR-324-5p expression. NEAT1 was up-regulated in TAA patients, as well as HAoSMC and HA-VSMC cells. Down-regulation of NEAT1 could inhibit the proliferative abilities while promoting apoptosis of TAA cells. MiR-324-5p expression was down-regulated in both TAA tissues and cells. Then, RAN was selected out as a target of miR-324-5p. More interestingly, miR-324-5p had inhibitory effects on malignant behaviors of TAA cells. RAN was negatively related with miR-324-5p while positively correlated with NEAT1 in the tissues. Finally, the data of rescue assays manifested that RAN up-regulation could countervail the influence of down-regulation of NEAT1 on TAA cells. NEAT1 could contribute to the malignant behaviors of TAA cells by targeting miR-324-5p/RAN. NEAT1 might be an underlying target for the therapy of TAA.

Abstract 13562: COQ8B : A Candidate Genetic Modifier of Thoracic Aortic Aneurysm Severity

Thoracic aortic aneurysm (TAA) is a latent aortopathy that predisposes to life-threatening aortic dissection. Personalized prediction of the risk for progressive aortic dilation and dissection is currently limited. The identification of genetic modifiers may facilitate more precise risk prediction and optimize clinical decisions. We previously identified COQ8B , a gene important for mitochondrial biosynthesis of coenzyme Q, as a candidate modifier of TAA severity. Biological evidence for a mitochondrial role in TAA pathogenesis includes severe mitochondrial ultrastructural defects in aortic smooth muscle cells (SMCs) and nitrated tyrosine residues in TAA tissues consistent with oxidative stress. In this study, we hypothesized that a common single nucleotide polymorphism (SNP) rs3865452 in COQ8B (c.521A&gt;G, p.H174R) modifies the rate of aortic root dilation. Forty-eight patients with TAA and a mean age at first echocardiogram of 10 ± 5 years were genotyped for rs3865452. Echocardiograms (mean 6.8 ± 4.0 per patient) were performed clinically over a mean interval of 5.3 ± 4.1 years. In mixed model analysis of longitudinal aortic root Z-scores, the c.521G allele was associated with a decreased rate of aortic root dilation in univariate analysis (p=0.0003) and multivariate analysis (p=0.03) that included sex and genetic syndrome as covariates. Hypothesizing a modifier role for COQ8B , we analyzed a separate cohort of 230 individuals with early onset thoracic aortic dissection. The c.521G allele was less frequent in dissection cases than controls (p=0.04), also consistent with a protective effect for this SNP. To better understand the mechanism of COQ8B effect in TAA, RNA levels of COQ8B in SMCs cultured directly from the aorta were assayed, demonstrating levels 2-fold lower in TAA compared with controls. siRNA knockdown of COQ8B in aortic SMCS resulted in decreased mitochondrial respiration, increased lipid peroxidation, and altered expression of SMC contractile genes. In conclusion, the findings of this study support a modifier role for the rs3865452 SNP in COQ8B in TAA, which may involve mitochondrial pathology and oxidative stress. This suggests that this SNP may be useful for clinical risk prediction.

A generic physics-informed neural network-based constitutive model for soft biological tissues

Constitutive modeling is a cornerstone for stress analysis of mechanical behaviors of biological soft tissues. Recently, it has been shown that machine learning (ML) techniques, trained by supervised learning, are powerful in building a direct linkage between input and output, which can be the strain and stress relation in constitutive modeling. In this study, we developed a novel generic physics-informed neural network material (NNMat) model which employs a hierarchical learning strategy by following the steps: (1) establishing constitutive laws to describe general characteristic behaviors of a class of materials; (2) determining constitutive parameters for an individual subject. A novel neural network structure was proposed which has two sets of parameters: (1) a class parameter set for characterizing the general elastic properties; and (2) a subject parameter set (three parameters) for describing individual material response. The trained NNMat model may be directly adopted for a different subject without re-training the class parameters, and only the subject parameters are considered as constitutive parameters. Skip connections are utilized in the neural network to facilitate hierarchical learning. A convexity constraint was imposed to the NNMat model to ensure that the constitutive model is physically relevant. The NNMat model was trained, cross-validated and tested using biaxial testing data of 63 ascending thoracic aortic aneurysm tissue samples, which was compared to expert-constructed models (Holzapfel-Gasser-Ogden, Gasser–Ogden–Holzapfel, and four-fiber families) using the same fitting and testing procedure. Our results demonstrated that the NNMat model has a significantly better performance in both fitting (R2 value of 0.9632 vs 0.9019, p=0.0053) and testing (R2 value of 0.9471 vs 0.8556, p=0.0203) than the Holzapfel–Gasser–Ogden model. The proposed NNMat model provides a convenient and general methodology for constitutive modeling.

Tissue-Specific miRNAs Regulate the Development of Thoracic Aortic Aneurysm: The Emerging Role of KLF4 Network.

MicroRNAs (miRNAs) are critical regulators of the functional pathways involved in the pathogenesis of cardiovascular diseases. Understanding of the disease-associated alterations in tissue and plasma will elucidate the roles of miRNA in modulation of gene expression throughout development of sporadic non-syndromic ascending thoracic aortic aneurysm (TAA). This will allow one to propose relevant biomarkers for diagnosis or new therapeutic targets for the treatment. The high-throughput sequencing revealed 20 and 17 TAA-specific miRNAs in tissue and plasma samples, respectively. qRT-PCR analysis in extended cohort revealed sex-related differences in miR-10a-5p, miR-126-3p, miR-155-5p and miR-148a-3p expression, which were the most significantly dysregulated in TAA tissues of male patients. Unexpectedly, the set of aneurysm-related miRNAs in TAA plasma did not resemble the tissue signature suggesting more complex organism response to the disease. Three of TAA-specific plasma miRNAs were found to be restored to normal level after aortic surgery, further signifying their relationship to the pathology. The panel of two plasma miRNAs, miR-122-3p, and miR-483-3p, could serve as a potential biomarker set (AUC = 0.84) for the ascending TAA. The miRNA-target enrichment analysis exposed TGF-β signaling pathway as sturdily affected by abnormally expressed miRNAs in the TAA tissue. Nearly half of TAA-specific miRNAs potentially regulate a key component in TGF-β signaling: TGF-β receptors, SMADs and KLF4. Indeed, using immunohistochemistry analysis we detected increased KLF4 expression in 27% of TAA cells compared to 10% of non-TAA cells. In addition, qRT-PCR demonstrated a significant upregulation of ALK1 mRNA expression in TAA tissues. Overall, these observations indicate that the alterations in miRNA expression are sex-dependent and play an essential role in TAA via TGF-β signaling.

MiR-574-5p: A Circulating Marker of Thoracic Aortic Aneurysm.

Thoracic aortic aneurysm (TAA) can lead to fatal complications such as aortic dissection. Since aneurysm dimension poorly predicts dissection risk, microRNAs (miRNAs) may be useful to diagnose or risk stratify TAA patients. We aim to identify miRNAs associated with TAA pathogenesis and that are possibly able to improve TAA diagnosis. MiRNA microarray experiments of aortic media tissue samples from 19 TAA patients and 19 controls allowed identifying 232 differentially expressed miRNAs. Using interaction networks between these miRNAs and 690 genes associated with TAA, we identified miR-574-5p as a potential contributor of TAA pathogenesis. Interestingly, miR-574-5p was significantly down-regulated in the TAA tissue compared to the controls, but was up-regulated in serum samples from a separate group of 28 TAA patients compared to 20 controls (p < 0.001). MiR-574-5p serum levels discriminated TAA patients from controls with an area under the receiver operating characteristic curve of 0.87. In the Fbn1C1041G/+ mouse model, miR-574-5p was down-regulated in aortic tissue compared to wild-type (p < 0.05), and up-regulated in plasma extracellular vesicles from Fbn1C1041G/+ mice compared to wild-type mice (p < 0.05). Furthermore, in vascular smooth muscle cells, angiotensin II appears to induce miR-574-5p secretion in extracellular vesicles. In conclusion, miR-574-5p is associated with TAA pathogenesis and may help in diagnosing this disease.

Mapping Semaphorins and Netrins in the Pathogenesis of Human Thoracic Aortic Aneurysms.

Thoracic aortic aneurysm (TAA) is a complex life-threatening disease characterized by extensive extracellular matrix (ECM) fragmentation and persistent inflammation, culminating in a weakened aorta. Although evidence suggests defective canonical signaling pathways in TAA, the full spectrum of mechanisms contributing to TAA is poorly understood, therefore limiting the scope of drug-based treatment. Here, we used a sensitive RNA sequencing approach to profile the transcriptomic atlas of human TAA. Pathway analysis revealed upregulation of key matrix-degrading enzymes and inflammation coincident with the axonal guidance pathway. We uncovered their novel association with TAA and focused on the expression of Semaphorins and Netrins. Comprehensive analysis of this pathway showed that several members were differentially expressed in TAA compared to controls. Immunohistochemistry revealed that Semaphorin4D and its receptor PlexinB1, similar to Netrin-1 proteins were highly expressed in damaged areas of TAA tissues but faintly detected in the vessel wall of non-diseased sections. It should be considered that the current study is limited by its sample size and the use of internal thoracic artery as control for TAA for the sequencing dataset. Our data determines important neuronal regulators of vascular inflammatory events and suggest Netrins and Semaphorins as potential key contributors of ECM degradation in TAA.

Characterization of SMAD2 Activation in Human Thoracic Aortic Aneurysm.

Objective: Thoracic aortic aneurysm (TAA) reflects the local expansion of the thoracic aorta; the underlying causal molecular mechanism of TAA is not well understood. Recent studies have shown the importance of transforming growth factor beta (TGFβ) signaling in Marfan and Loeys–Dietz syndromes; however, its role in non-familial, non-syndromic TAA remains unclear.Materials and Methods: We performed histochemical and immunohistochemical analyses for activated (phosphorylated) SMAD2 (P-SMAD2) as an indicator of TGFβ signaling activities in the ascending TAA tissue as well as in the ascending aortic tissue with a normal diameter obtained from 7 patients without any clinical findings suggesting familial or syndromic TAA.Results: TAA samples showed a higher P-SMAD2-positive area than samples with a normal diameter. P-SMAD2 signal was higher in the outer zone of the aortic and TAA walls. Within the TAA tissue, P-SMAD2 staining showed the following two distinct patterns: layer-like staining at the border of the medial layer and the thickened intima and a spot-like staining within the medial layer surrounding the microvessels.Conclusion: These findings suggested that TGFβ signaling is activated in several distinct histopathological contexts in TAA, suggesting a complex role of TGFβ.

MiRNome Profiling in Bicuspid Aortic Valve-Associated Aortopathy by Next-Generation Sequencing.

The molecular mechanisms underlying thoracic aortic aneurysm (TAA) in patients with bicuspid aortic valve (BAV) are incompletely characterized. MicroRNAs (miRNAs) may play a major role in the different pathogenesis of aortopathy. We sought to employ next-generation sequencing to analyze the entire miRNome in TAA tissue from patients with BAV and tricuspid aortic valve (TAV). In the discovery stage, small RNA sequencing was performed using the Illumina MiSeq platform in 13 TAA tissue samples (seven patients with BAV and six with TAV). Gene ontology (GO) and KEGG pathway analysis were used to identify key pathways and biological functions. Validation analysis was performed by qRT-PCR in an independent cohort of 30 patients with BAV (26 males; 59.5 ± 12 years) and 30 patients with TAV (16 males; 68.5 ± 9.5 years). Bioinformatic analysis identified a total of 489 known mature miRNAs and five novel miRNAs. Compared to TAV samples, 12 known miRNAs were found to be differentially expressed in BAV, including two up-regulated and 10 down-regulated (FDR-adjusted p-value ≤ 0.05 and fold change ≥ 1.5). GO and KEGG pathway enrichment analysis (FDR-adjusted p-value < 0.05) identified different target genes and pathways linked to BAV and aneurysm formation, including Hippo signaling pathway, ErbB signaling, TGF-beta signaling and focal adhesion. Validation analysis of selected miRNAs confirmed the significant down-regulation of miR-424-3p (p = 0.01) and miR-3688-3p (p = 0.03) in BAV patients as compared to TAV patients. Our study provided the first in-depth screening of the whole miRNome in TAA specimens and identified specific dysregulated miRNAs in BAV patients.

A reproducible swine model of proximal descending thoracic aortic aneurysm created with intra-adventitial application of elastase

ObjectiveAnimal models are required to explore the mechanisms of and therapy for proximal descending thoracic aortic aneurysm (TAA). This study aimed to establish a reproducible swine model of proximal descending TAA that can further explain the occurrence and progression of proximal descending TAA. MethodsEighteen Chinese Wuzhishan miniature pigs (30.32 ± 1.34 kg) were randomized into the elastase group (n = 12) and the control group (n = 6). The elastase group received intra-adventitial injections of elastase (5 mL, 20 mg/mL), and the control group received injections of physiologic saline solution. A 4-cm descending thoracic aortic segment proximal to the left subclavian artery was isolated. The distance between the left subclavian artery and the injection starting point of the descending thoracic aorta was 0.5 cm. Elastic protease was circumferentially injected intra-adventitially into the isolated segment of the aortic wall in the elastase group by a handmade bent syringe. The length of the elastic protease injection was 2 cm. An average of 12 injection points were distributed in this 2-cm aortic segment. Each injection point used about 0.4 mL of elastic protease. The distance between two injection points was about 1.5 cm. All animals underwent digital subtraction angiography preoperatively and 3 weeks after operation. Three weeks after TAA induction, aortas were harvested for biochemical and histologic measurements. ResultsAll animals in the elastase group developed TAAs. No aneurysms were observed in the control group. The distance between the left subclavian artery and the TAA was 8.00 ± 4.19 mm. Preoperative and postoperative aortic diameters of the elastase group were 15.42 ± 0.43 mm and 24.53 ± 1.41 mm, respectively (P < .0001). Preoperative and postoperative aortic diameters of the control group were 15.31 ± 0.33 mm and 15.57 ± 0.40 mm, respectively (P = .5211). The changes of aortic structure and composition included reduction of smooth muscle cells and degradation of elastic fibers. Levels of matrix metalloproteinases 2 and 9 were increased in TAA tissue. ConclusionsThis study established a reproducible large animal model of proximal descending TAA. This model has the same biochemical characteristics as human aneurysms in the aspects of aortic expansion, aortic middle-level degeneration, and changes in the levels of matrix metalloproteinases and provides a platform for further study.

Abstract 135: Thoracic Aortic Wall Tension Regulates microRNA-133a Abundance

Background: MicroRNA-133a (miR133a) is a small non-coding RNA, which represses the translation of multiple mRNAs. This laboratory has reported an inverse relationship between aortic diameter and miR133a abundance in aortic tissue from patients with thoracic aortic aneurysm (TAA); as diameter increased, the abundance of miR133a decreased. Given that wall tension at a given pressure increases with increasing vessel diameter (Law of LaPlace), this study tested the hypothesis that elevated aortic wall tension results in a loss of miR-133a. Methods/Results: TAA was induced in wild type mice using an established murine model (0.5M CaCl 2 application, 15 min). MiR133a abundance (QPCR) was reduced in TAA tissue (3-wk TAA, 42.1±8.6% p&lt;0.05 vs mice without TAA (100%)). In two in vivo models of elevated wall tension ( simulated hypertension ): 1) ANGII (angiotensin II infusion; 1.44mg/kg/day), and 2) BPH2 (spontaneously hypertensive mice, The Jackson Laboratory, Stock #003005), miR133a levels were decreased compared to normotensive controls (ANGII: 53.0±4.3%; BPH2: 51.7±7.0%; p&lt;0.05 vs normotensive control (100%)). Aortic rings from wild type mice were hung on parallel wires in an ex vivo tissue myograph at 0.7 g, then ANGII (100nM) was added to the tissue baths, which generated increased tension (1.21±0.15g) and resulted in reduced tissue miR133a abundance (46.0±12%; p&lt;0.05 vs no AngII,). Furthermore, increased tension alone (1.5g, 3 hr) resulted in decreased tissue miR133a abundance (39.0±7.0%; p&lt;0.05 vs 0.7 g tension). Isolated primary aortic cell lines (fibroblasts (FB) and smooth muscle cells (SMC)) were exposed to biaxial cyclic stretch for 3 hr. FB miR133a was reduced (62.8±8.3%; p&lt;0.05 vs unstretched control (100%)), while SMC miR133a abundance remained unchanged. Conclusion: The significance of these unique findings is 2-fold: First, tension alone was sufficient to decrease miR133a abundance in aortic tissue. Second, increased tension reduced miR133a abundance in FB, a cell type that is responsible for extracellular matrix remodeling. These findings suggest changes in wall tension alone ( hypertension ) may be associated with pathological extracellular matrix remodeling, in part, through the loss of miR133a in fibroblasts.

Abstract 467: PCSK6 Is Upregulated in Vascular Diseases Characterized by Inflammation and Smooth Muscle Cell Proliferation

Recently we demonstrated upregulation of the proprotein convertase PCSK6 in a large cohort of human carotid atherosclerotic plaques (n=127) compared to normal arteries and in symptomatic vs. asymptomatic lesions. PCSK6 was localized to smooth muscle cells (SMCs) in the fibrous cap and showed a positive correlation to markers of inflammation, extracellular matrix remodeling and cytokines. Here, we aimed at elucidating the role of this protease in vascular disease by examining its expression in different human pathologies and in animal models. Increased expression of PCSK6 in vascular diseases was validated in public microarray datasets and other available human cohorts. PCSK6 was upregulated in carotid atherosclerotic plaques vs. controls (n=32 patients, p&lt;0.0001), as well as in abdominal aortic aneurysm (AAA) vs. normal tissue (n=14, p&lt;0.0001) and in thoracic aortic aneurysm (TAA) tissue from bicuspid vs. tricuspid patients (n=244, p=0.012). By eQTL analyses, several SNPs in the PCSK6 genomic region were shown to influence its expression in carotid plaques and TAA tissue. Among these, rs6598465 showed a mild association to progression of maximum intima-media thickness in the left and right arteries in a separate cohort of high-risk coronary artery disease subjects (n=3400, p=0.037). By immunohistochemistry, PCSK6 localized mainly to SMCs in carotid plaques, AAA and TAA tissue, but was also found to be expressed by CD68 and CD163+ macrophages. Investigation of mouse, rat and human artery tissues with pronounced intimal hyperplasia revealed strong expression of PCSK6 in proliferating SMCs. In rat carotid artery balloon injury, PCSK6 was downregulated in the early phases after injury mostly defined by inflammatory response, while upregulated in later phases with prominent SMC activation, and consistently localized in SMCs. Expression of PCSK6 in this model was strongly positively correlated solely to PDGFB and IGF1 (p&lt;0.0001), cytokines known to induce SMC proliferation. We established a functional link between elevated expression of PCSK6 and vascular diseases characterized by inflammation and SMC proliferation. Further investigations in vitro are necessary to provide mechanistic insight into the role of this protease in vascular disease.

Abstract 291: Upregulation of Multiple DNA Methyltransferase Isoforms is Associated With Tissue Inflammation in Human Thoracic Aortic Aneurysm

Thoracic aortic aneurysm (TAA) is a degenerative condition of the aorta characterized by aortic dilatation secondary to tissue inflammation and matrix remodeling, which ultimately progresses to aortic dissection and rupture. While recent work suggests that in vitro exposure to pro-inflammatory stimuli promotes DNMT3b-dependent DNA methylation in skeletal muscle, epigenetic mechanisms regulating gene expression in TAA remain largely uncharacterized. We therefore tested the hypothesis that expression of tumor necrosis factor α (TNF-α) is increased in human TAA tissue and that such increases are associated with upregulation of DNA methyltransferase (DNMT) levels. Normal aortic samples (n=13) were acquired from non-aneurysmal hearts/aorta not suitable for cardiac transplant, whereas TAA samples from patients with tricuspid aortic valves (n=29) were acquired from patients undergoing elective surgery for aortic aneurysm. We measured relative expression of TNF-α, DNMT1, DNMT3a and DNMT3b mRNA levels in normal and TAA tissue by qRT-PCR, and DNMT3b protein levels by western blot. Marked increases in expression of the inflammatory marker TNF-α were noted in TAA tissue (12.3± 2.34), and were associated with significantly increased DNMT1 (1.4±0.13), DNMT3a (1.44±0.09) and DNMT3b (1.94±0.27) mRNA levels (p&lt;0.05 for all). DNMT3b protein levels were also dramatically increased in human TAA samples (2.99±0.58-fold, n = 10/group, p&lt;0.05) when compared to normal aortic tissue. Collectively, these data suggest a novel potential role for DNA methyltransferases as major regulators of altered gene expression in TAA, and support our working hypothesis that inflammation-induced increases in DNMT3b levels may be critical repressors of protective gene expression in TAA.