Vascular dysfunction programmed in male rats by topiramate during peripubertal period.

AIM: The present study evaluated whether topiramate (TPM) treatment during the peripubertal period affects vascular parameters of male rats and whether oxidative stress plays a role in these changes. MAIN METHODS: Rats were treated with TPM (41 mg/kg/day, gavage) or vehicle (CTR group) from the postnatal day (PND) 28 to 50. At PND 51 and 120 the rats were evaluated for: thoracic aorta reactivity to phenylephrine, in the presence (Endo+) or absence of endothelium (Endo-), to acetylcholine and to sodium nitroprusside (SNP), aortic thickness and endothelial nitric oxide synthase (eNOS) expression. In serum were analyzed: the antioxidant capacity by ferric reducing antioxidant power assay; endogenous antioxidant reduced glutathione, and superoxide anion. Results were expressed as mean ± s.e.m., differences when p < 0.05. STATISTICS: Two-way ANOVA (and Tukey's) or Student t-test. KEY FINDINGS: At PND 51, the contraction induced by phenylephrine in Endo+ ring was higher in TPM when compared to CTR. At PND 120, the aortic sensitivity to acetylcholine in TPM rats was reduced in comparison with CTR. The aortic eNOs expression and the aortic thickness were similar between the groups. At PND 51 and 120, TPM group presented a decrease in antioxidants when compared to CTR groups and at PND 120, in TPM group the superoxide anion was increased. SIGNIFICANCE: Taken together, the treatment of rats with TPM during peripubertal period promoted permanent impairment of endothelial function probably mediated by oxidative stress.

A cell atlas of thoracic aortic perivascular adipose tissue: a focus on mechanotransducers.

Perivascular adipose tissue (PVAT) is increasingly recognized for its function in mechanotransduction. However, major gaps remain in our understanding of the cells present in PVAT, as well as how different cells contribute to mechanotransduction. We hypothesized that snRNA-seq would reveal the expression of mechanotransducers, and test one (PIEZO1) to illustrate the expression and functional agreement between single-nuclei RNA sequencing (snRNA-seq) and physiological measurements. To contrast two brown tissues, subscapular brown adipose tissue (BAT) was also examined. We used snRNA-seq of the thoracic aorta PVAT (taPVAT) and BAT from male Dahl salt-sensitive (Dahl SS) rats to investigate cell-specific expression mechanotransducers. Localization and function of the mechanostransducer PIEZO1 were further examined using immunohistochemistry (IHC) and RNAscope, as well as pharmacological antagonism. Approximately 30,000 nuclei from taPVAT and BAT each were characterized by snRNA-seq, identifying eight major cell types expected and one unexpected (nuclei with oligodendrocyte marker genes). Cell-specific differential gene expression analysis between taPVAT and BAT identified up to 511 genes (adipocytes) with many (≥20%) being unique to individual cell types. Piezo1 was the most highly, widely expressed mechanotransducer. The presence of PIEZO1 in the PVAT but not the adventitia was confirmed by RNAscope and IHC in male and female rats. Importantly, antagonism of PIEZO1 by GsMTX4 impaired the PVAT's ability to hold tension. Collectively, the cell compositions of taPVAT and BAT are highly similar, and PIEZO1 is likely a mechanotransducer in taPVAT.NEW & NOTEWORTHY This study describes the atlas of cells in the thoracic aorta perivascular adipose tissue (taPVAT) of the Dahl-SS rat, an important hypertension model. We show that mechanotransducers are widely expressed in these cells. Moreover, PIEZO1 expression is shown to be restricted to the taPVAT and is functionally implicated in stress relaxation. These data will serve as the foundation for future studies investigating the role of taPVAT in this model of hypertensive disease.

Effects of the Heterodimeric Neurotoxic Phospholipase A2 from the Venom of Vipera nikolskii on the Contractility of Rat Papillary Muscles and Thoracic Aortas.

Phospholipases A2 (PLA2s) are a large family of snake toxins manifesting diverse biological effects, which are not always related to phospholipolytic activity. Snake venom PLA2s (svPLA2s) are extracellular proteins with a molecular mass of 13-14 kDa. They are present in venoms in the form of monomers, dimers, and larger oligomers. The cardiovascular system is one of the multiple svPLA2 targets in prey organisms. The results obtained previously on the cardiovascular effects of monomeric svPLA2s were inconsistent, while the data on the dimeric svPLA2 crotoxin from the rattlesnake Crotalus durissus terrificus showed that it significantly reduced the contractile force of guinea pig hearts. Here, we studied the effects of the heterodimeric svPLA2 HDP-1 from the viper Vipera nikolskii on papillary muscle (PM) contractility and the tension of the aortic rings (ARs). HDP-1 is structurally different from crotoxin, and over a wide range of concentrations, it produced a long-term, stable, positive inotropic effect in PMs, which did not turn into contractures at the concentrations studied. This also distinguishes HDP-1 from the monomeric svPLA2s, which at high concentrations inhibited cardiac function. HDP-1, when acting on ARs preconstricted with 10 µM phenylephrine, induced a vasorelaxant effect, similar to some other svPLA2s. These are the first indications of the cardiac and vascular effects of true vipers' heterodimeric svPLA2s.

Vasorelaxant effects of ellagitannins isolated from Cuphea carthagenensis.

Cuphea carthagenensis (Jacq.) J. F. Macbr. is a popular plant in Brazilian folk medicine owing to its hypotensive and central nervous system depressant effects. This study aimed to validate the hypotensive effect of the plant's aqueous extract (AE) in rats and examine the vascular actions of three hydrolyzable tannins, oenothein B, woodfordin C, and eucalbanin B, isolated from AE. Systolic blood pressure in unanesthetized rats was determined using the non-invasive tail-cuff method. Oral treatment of normotensive rats with 0.5 and 1.0 g/kg/day AE induced a dose-related hypotensive effect after 1 week. In rat aortic rings pre-contracted with noradrenaline, all ellagitannins (20 - 180 µM) induced a concentration-related vasorelaxation. This effect was blocked by either removing the endothelium or pre-incubating with NG-nitro-l-arginine methyl ester (10 µM), an inhibitor of nitric oxide (NO) synthase. In KCl-depolarized rat portal vein preparations, the investigated compounds did not affect significantly the maximal contractile responses and pD2 values of the concentration-response curves to CaCl2. Our results demonstrated the hypotensive effect of C. carthagenensis AE in unanesthetized rats. All isolated ellagitannins induced vasorelaxation in vitro via activating NO synthesis/NO release from endothelial cells, without altering the Ca2+ influx in vascular smooth muscle preparations. Considering the low oral bioavailability of ellagitannins, the determined in vitro actions of these compounds are unlikely to account for the hypotensive effect of AE in vivo. It remains to be determined the role of the bioactive ellagitannin-derived metabolites in the hypotensive effect observed after oral treatment of unanesthetized rats with the plant extract.

Inhibition of heme oxygenase 1 alleviates thoracic aortic aneurysm via restoration of extracellular matrix.

BACKGROUND: Thoracic aortic aneurysm (TAA) is a silent but life-threatening cardiovascular disease. Heme oxygenase 1 (HO-1) plays an important role in the cardiovascular diseases but is poorly understood in TAA. This study aims at investigating the role of HO-1 in TAA. METHODS: Single-cell RNA sequencing, Western blot and histological assay were performed to identify specific cellular expression of HO-1 in both human and ß-aminopropionitrile (BAPN)-induced mice TAA. Zinc protoporphyrin (ZnPP), a pharmacological inhibitor of HO-1, was used to investigate whether inhibition of HO-1 could attenuate BAPN-induced TAA in rodent model. Histological assay, Western blot assay, and mRNA sequencing were further performed to explore the underlying mechanisms. RESULTS: Single-cell transcriptomic analyses of 113,800 thoracic aortic cells identified an increase of HO-1(+) macrophage in aneurysmal thoracic aorta from BAPN-induced TAA mice and TAA patients. Histological assay verified HO-1 overexpression in clinical TAA specimens, which was co-localized with CD68(+) macrophage. HO-1(+) macrophage was closely associated with pro-inflammatory response and immune activation. Inhibition of HO-1 through ZnPP significantly alleviated BAPN-induced TAA in mice and restored extracellular matrix (ECM) in vivo. Further experiments showed that ZnPP treatment suppressed the expression of matrix metalloproteinases (MMPs) in aneurysmal thoracic aortic tissues from BAPN-induced TAA mice, including MMP2 and MMP9. Macrophages from myeloid specific HO-1 knockout mice displayed weakened pro-inflammatory activity and ECM degradation capability. CONCLUSION: HO-1(+) macrophage subgroup is a typical hallmark of TAA. Inhibition of HO-1 through ZnPP alleviates BAPN-induced TAA in mice, which might work through restoration of ECM via suppressing MMP2 and MMP9 expression.

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.

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

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

The Role of ADAMTS Proteoglycanases in Thoracic Aortic Disease.

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

Aortic regurgitation provokes phenotypic modulation of smooth muscle cells in the normal ascending aorta.

BACKGROUND: Aortic complications are more likely to occur in patients with ascending aortic aneurysms and concomitant aortic regurgitation (AR). AR may have a negative influence on the aortic wall structure even in patients with tricuspid aortic valves and absence of aortic dilatation. It is unknown whether smooth muscle cell (SMC) changes are a feature of AR-associated aortic remodeling. METHODS: Nondilated aortic samples were harvested intraoperatively from individuals with normal aortic valves (n = 10) or those with either predominant aortic stenosis (AS) (n = 20) or AR (n = 35). Tissue from each patient was processed for immunohistochemistry or used for the extraction of medial SMCs. Tissue and cells were stained for markers of SMC contraction (alpha-smooth muscle actin), synthesis (vimentin) and senescence (p16INK4A and p21Cip1 [p16/p21]). Replicative capacity was analyzed in cultured SMCs from AS- and AR-associated aortas. A subanalysis compared SMCs from individuals with either tricuspid aortic valves or bicuspid aortic valves to evaluate the effect of aortic valve morphology. RESULTS: In aortic tissue samples, AR was associated with decreased alpha-smooth muscle actin and increased vimentin, p16 and p21 compared with normal aortic valves and AS. In cell culture, SMCs from AR-aortas had decreased alpha-smooth muscle actin and increased vimentin compared with SMCs from AS-aortas. AR-associated SMCs had increased p16 and p21 expression, and they reached senescence earlier than SMCs from AS-aortas. In AR, SMC changes were more pronounced with the presence of a bicuspid aortic valve. CONCLUSIONS: AR itself negatively influences SMC phenotype in the ascending aortic wall. This AR-specific effect is independent of aortic diameter and aortic valve morphology, although it is more pronounced with bicuspid aortic valves. These findings provide insight into the mechanisms of AR-related aortic remodeling, and they provide a model for studying SMC-specific therapies in culture.

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

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

Estrogen ameliorates sepsis-induced vascular hyporeactivity in thoracic aorta of female rats via permissive effect of GRα expression.

OBJECTIVE: Estrogen is correlated to the lower mortality and disease severity of female than that of male, which indicates the potential therapeutic role of estrogen supplement therapy in sepsis. The structure of Daidzein is similar to that of 17ß estradiol (E2), an estrogen in human body, causing the exogenous Daidzein can interact with estrogen receptor as well as E2 in the body. We aim to explore the therapeutic role of estrogen in sepsis-induced vascular dysfunction. Also, we wonder if estrogen regulates blood pressure via glucocorticoid-mediated vascular reactivity. METHODS: Female SD rats received ovariectomy (OVX) to induce estrogen deficiency. After 12 weeks of administration, cecal ligation and puncture (CLP) was used to establish the in vivo model of sepsis. Lipopolysaccharide (LPS) was used to construct the in vitro model of sepsis in vascular smooth muscle cells (VSMCs). E2 and Daidzein were used for estrogen supplement therapy. RESULTS: E2 and Daidzein significantly inhibited inflammation infiltration and histopathological injury in thoracic aorta in the rat model with CLP. E2 and Daidzein improved carotid pressure and vascular hyporeactivity in sepsis rats with OVX. Importantly, E2 and Daidzein promoted glucocorticoid permissive action and increased glucocorticoid receptor α (GRα) expression in thoracic aorta smooth muscle cells. E2 and Daidzein upregulated GRα, and inhibits cytokine production, proliferative phenotype and cell migration in LPS-induced VSMCs. CONCLUSION: Estrogen improved vascular hyporeactivity in thoracic aorta induced by sepsis via permissive effect of GRα expression.

Perivascular adipose tissue promotes vascular dysfunction in murine lupus.

Introduction: Patients with systemic lupus erythematosus (SLE) are at elevated risk for Q10 cardiovascular disease (CVD) due to accelerated atherosclerosis. Compared to heathy control subjects, lupus patients have higher volumes and densities of thoracic aortic perivascular adipose tissue (PVAT), which independently associates with vascular calcification, a marker of subclinical atherosclerosis. However, the biological and functional role of PVAT in SLE has not been directly investigated. Methods: Using mouse models of lupus, we studied the phenotype and function of PVAT, and the mechanisms linking PVAT and vascular dysfunction in lupus disease. Results and discussion: Lupus mice were hypermetabolic and exhibited partial lipodystrophy, with sparing of thoracic aortic PVAT. Using wire myography, we found that mice with active lupus exhibited impaired endothelium-dependent relaxation of thoracic aorta, which was further exacerbated in the presence of thoracic aortic PVAT. Interestingly, PVAT from lupus mice exhibited phenotypic switching, as evidenced by "whitening" and hypertrophy of perivascular adipocytes along with immune cell infiltration, in association with adventitial hyperplasia. In addition, expression of UCP1, a brown/beige adipose marker, was dramatically decreased, while CD45-positive leukocyte infiltration was increased, in PVAT from lupus mice. Furthermore, PVAT from lupus mice exhibited a marked decrease in adipogenic gene expression, concomitant with increased pro-inflammatory adipocytokine and leukocyte marker expression. Taken together, these results suggest that dysfunctional, inflamed PVAT may contribute to vascular disease in lupus.

Pyridoxamine prevents increased atherosclerosis by intermittent methylglyoxal spikes in the aortic arches of ApoE-/- mice.

Methylglyoxal (MGO) is a reactive glucose metabolite linked to diabetic cardiovascular disease (CVD). MGO levels surge during intermittent hyperglycemia. We hypothesize that these MGO spikes contribute to atherosclerosis, and that pyridoxamine as a MGO quencher prevents this injury. To study this, we intravenously injected normoglycemic 8-week old male C57Bl6 ApoE-/- mice with normal saline (NS, n = 10) or 25 µg MGO for 10 consecutive weeks (MGOiv, n = 11) with or without 1 g/L pyridoxamine (MGOiv+PD, n = 11) in the drinking water. We measured circulating immune cells by flow cytometry. We quantified aortic arch lesion area in aortic roots after Sudan-black staining. We quantified the expression of inflammatory genes in the aorta by qPCR. Intermittent MGO spikes weekly increased atherosclerotic burden in the arch 1.8-fold (NS: 0.9 ± 0.1 vs 1.6 ± 0.2 %), and this was prevented by pyridoxamine (0.8 ± 0.1 %). MGOiv spikes increased circulating neutrophils and monocytes (2-fold relative to NS) and the expression of ICAM (3-fold), RAGE (5-fold), S100A9 (2-fold) and MCP1 (2-fold). All these changes were attenuated by pyridoxamine. This study suggests that MGO spikes damages the vasculature independently of plasma glucose levels. Pyridoxamine and potentially other approaches to reduce MGO may prevent excess cardiovascular risk in diabetes.

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.

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

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

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.

Prenatal hypothyroidism diminished exogenous NO-mediated diastolic effects in fetal rat thoracic aorta smooth muscle via increased oxidative stress.

Maternal hypothyroidism is an important problem of modern healthcare and is reported to increase the risk of cardiovascular diseases in the offspring later in life. However, it is unclear whether hypothyroidism during pregnancy causes vascular damage in the fetal period. We established the prenatal hypothyroidism rat model and collected the fetuses at the 21th day of gestation (GD21). Thyroid hormone concentrations in maternal and offspring blood serum were assessed by enzyme-linked immunosorbent assay (ELISA). The thoracic aortas of the fetuses were isolated for microvessel functional testing and histochemical stainings. qPCR and Western blot were performed to access mRNA and protein expression. We found that the concentrations of thyroid hormones in the serum of pregnant rats and fetuses were significantly suppressed at GD21. The responses of the fetal thoracic aortas to SNP were significantly attenuated in the PTU group. However, no statistical difference was found between the two groups when treated with either inhibitor (ODQ) or activator (BAY58-2667) of sGC. The production of O2-• in the arterial wall was significantly increased in hypothyroid fetuses. Moreover, the level of NADPH oxidase (NOX) was increased, while superoxide dismutase 2 (SOD2) was down-regulated in the PTU group, ultimately contributing to the increased production of superoxide. Additionally, decreased SNP-mediated vasodilation found in fetal vessels was improved by either NOX inhibitor (Apocynin) or SOD mimic (Tempol). These results indicate that increased oxidative stress is probably the cause of the diminished diastolic effect of exogenous NO in the thoracic artery of prenatal hypothyroidism exposed fetuses.

Rosa damascena Miller essential oil relaxes rat thoracic aorta through the NO-cGMP-dependent pathway.

AIM: This study aimed to investigate the effects of Rosa damascena Mill. essential oil on the vascular activity of rat thoracic aorta and its underlying mechanisms. METHODS: Experiments were performed using the isolated tissue bath model and Wistar rats. 0.1, 1, 10, and 100 µg/mL concentrations of rose oil were administered in all groups. To determine the vasoactive effects of rose oil, submaximal contractions were conducted by applying 10-5 M PE and 45 mM KCl separately in both endothelium-intact and -denuded segments. Time-matched distilled water groups were formed for control. To evaluate the role of endothelium-derived vasodilative factors, endothelium-intact segments were incubated with nitric oxide synthase inhibitor L-NAME, soluble guanylate cyclase inhibitor ODQ, and a non-selective cyclooxygenase inhibitor INDO. The statistical significance level was considered as p < 0.05. RESULTS: 1, 10, and 100 µg/mL rose oil doses led to vasorelaxation in thoracic aortas precontracted with 10-5 M PE (p: 0.029, p: 0.000, p: 0.000, respectively). In precontracted thoracic aortas with 45 mM KCl, the significant effect of rose oil persisted, albeit slightly diminished. When the endothelium was removed, the relaxant effect of rose oil was partially reduced, but still significant (p: 0.035, p: 0.028, p: 0.000, respectively). Preincubations with L-NAME and ODQ significantly attenuated rose oil-induced relaxation of endothelium-intact aortas precontracted with 10-5 M PE. In contrast, preincubation INDO did not modulate rose oil-induced relaxation. CONCLUSION: In conclusion, it was shown for the first time that rose oil can significantly mediate vasorelaxation in both PE and KCl precontracted rat thoracic aortas. Rose oil induced vasodilation with or without endothelium in a concentration-dependent manner. It was also shown that rose oil-induced vasorelaxant effects were reduced by L-NAME or ODQ pretreatment, but not modulated by INDO. These results demonstrated that rose oil-induced endothelium-dependent vasodilation is mediated by the NO-cGMP-dependent pathway.

Vascular Functional Effect Mechanisms of Elabela in Rat Thoracic Aorta.

BACKGROUND: Elabela is a recently discovered peptide hormone. The present study aims to investigate the vasorelaxant effect mechanisms of elabela in the rat thoracic aorta. METHODS: The vascular rings obtained from the thoracic aortas of the male Wistar albino rats were placed in the isolated tissue bath system. Resting tension was set to 1 gram. After the equilibration period, the vessel rings were contracted with phenylephrine or potassium chloride. Once a stable contraction was achieved, elabela-32 was applied cumulatively (10-9-10-6 molar) to the vascular rings. The experimental protocol was repeated in the presence of specific signaling pathway inhibitors or potassium channel blockers to determine the effect mechanisms of elabela. RESULTS: Elabela showed a significant vasorelaxant effect in a concentration-dependent manner (P < 0.001). The vasorelaxant effect level of elabela was significantly reduced by the apelin receptor antagonist F13A, cyclooxygenase inhibitor indomethacin, adenosine monophosphate-activated protein kinase inhibitor dorsomorphin, protein kinase C inhibitor bisindolmaleimide, large-conductance calcium-activated potassium channel blocker iberiotoxin, and intermediate-conductance calcium-activated potassium channel blocker TRAM-34 (P < 0.001). However, the vasorelaxant effect level of elabela was not significantly affected by the endothelial nitric oxide synthase inhibitor nitro-L-arginine methyl ester and mitogen-activated protein kinase inhibitor U0126. CONCLUSIONS: Elabela exhibits a prominent vasodilator effect in rat thoracic aorta. Apelin receptor, prostanoids, adenosine monophosphate-activated protein kinase, protein kinase C, and calcium-activated potassium channels are involved in the vasorelaxant effect mechanisms of elabela.

The role of biglycan in the healthy and thoracic aneurysmal aorta.

The class I small leucine-rich proteoglycan biglycan is a crucial structural extracellular matrix component that interacts with a wide range of extracellular matrix molecules. In addition, biglycan is involved in sequestering growth factors such as transforming growth factor-ß and bone morphogenetic proteins and thereby regulating pathway activity. Biglycan consists of a 42-kDa core protein linked to two glycosaminoglycan side chains and both are involved in protein interactions. Biglycan is encoded by the BGN gene located on the X-chromosome and is expressed in various tissues, including vascular tissue, skin, brain, kidney, lung, the immune system, and the musculoskeletal system. Although an increasing amount of data on the biological function of biglycan in the vasculature has been produced, its role in thoracic aortic aneurysms is still not fully elucidated. This review focuses on the role of biglycan in the healthy thoracic aorta and the development of thoracic aortic aneurysm and dissections in both mice and humans.