Smooth muscle cell phenotypic switching occurs independent of aortic dilation in bicuspid aortic valve-associated ascending aortas.

BACKGROUND: Bicuspid aortic valves (BAV) are frequently associated with ascending aortic aneurysms. The etiology is incompletely understood, but genetic factors, in addition to flow perturbations, are likely involved. Since loss of contractility and elaboration of extracellular matrix in the vessel wall are features of BAV-associated aortopathy, phenotypic modulation of smooth muscle cells (SMCs) may play a role. METHODS: Ascending aortic tissue was collected intra-operatively from 25 individuals with normal (i.e., tricuspid) aortic valves (TAV) and from 25 individuals with BAVs. For both TAV and BAV, 10 patients had non-dilated (ND) and 15 patients had dilated (D) aortas. SMCs were isolated and cultured from a subset of patients from each group. Aortic tissue and SMCs were fluorescently immunolabeled for SMC phenotypic markers (i.e., alpha-smooth muscle actin (ASMA, contractile), vimentin (synthetic) and p16INK4a and p21Cip1 (senescence). SMCs were also analyzed for replicative senescence in culture. RESULTS: In normal-sized and dilated BAV aortas, SMCs switched from the contractile state to either synthetic or senescent phenotypes, as observed by loss of ASMA (ND: P = 0.001, D: P = 0.002) and associated increases in vimentin (ND: P = 0.03, D: P = 0.004) or p16/p21 (ND: P = 0.03, D: P<0.0001) compared to TAV. Dilatation of the aorta exacerbated SMC phenotypic switching in both BAV and TAV aortas (all P<0.05). In SMCs cultured from normal and dilated aortas, those isolated from BAV reached replicative senescence faster than those from TAV aortas (all P = 0.02). Furthermore, there was a stark inverse correlation between ASMA and cell passage number in BAV SMCs (ND: P = 0.0006, D: P = 0.01), but not in TAV SMCs (ND: P = 0.93, D: P = 0.20). CONCLUSIONS: The findings of this study provide direct evidence from cell culture studies implying that SMCs switch from the contractile state to either synthetic or senescent phenotypes in the non-dilated BAV aorta. In cultured SMCs from both non-dilated and dilated aortas, we found that this process may precede dilatation and accompany aneurysm development in BAV. Our findings suggest that therapeutically targeting SMC phenotypic modulation in BAV patients may be a viable option to prevent or delay ascending aortic aneurysm formation.

Spatial transcriptomics reveals novel genes during the remodelling of the embryonic human arterial valves.

Abnormalities of the arterial valves, including bicuspid aortic valve (BAV) are amongst the most common congenital defects and are a significant cause of morbidity as well as predisposition to disease in later life. Despite this, and compounded by their small size and relative inaccessibility, there is still much to understand about how the arterial valves form and remodel during embryogenesis, both at the morphological and genetic level. Here we set out to address this in human embryos, using Spatial Transcriptomics (ST). We show that ST can be used to investigate the transcriptome of the developing arterial valves, circumventing the problems of accurately dissecting out these tiny structures from the developing embryo. We show that the transcriptome of CS16 and CS19 arterial valves overlap considerably, despite being several days apart in terms of human gestation, and that expression data confirm that the great majority of the most differentially expressed genes are valve-specific. Moreover, we show that the transcriptome of the human arterial valves overlaps with that of mouse atrioventricular valves from a range of gestations, validating our dataset but also highlighting novel genes, including four that are not found in the mouse genome and have not previously been linked to valve development. Importantly, our data suggests that valve transcriptomes are under-represented when using commonly used databases to filter for genes important in cardiac development; this means that causative variants in valve-related genes may be excluded during filtering for genomic data analyses for, for example, BAV. Finally, we highlight "novel" pathways that likely play important roles in arterial valve development, showing that mouse knockouts of RBP1 have arterial valve defects. Thus, this study has confirmed the utility of ST for studies of the developing heart valves and broadens our knowledge of the genes and signalling pathways important in human valve development.

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.

Bicuspid aortic valve aortopathy is characterized by embryonic epithelial to mesenchymal transition and endothelial instability.

Bicuspid aortic valve (BAV) is the most common congenital heart malformation frequently associated with ascending aortic aneurysm (AscAA). Epithelial to mesenchymal transition (EMT) may play a role in BAV-associated AscAA. The aim of the study was to investigate the type of EMT associated with BAV aortopathy using patients with a tricuspid aortic valve (TAV) as a reference. The state of the endothelium was further evaluated. Aortic biopsies were taken from patients undergoing open-heart surgery. Aortic intima/media miRNA and gene expression was analyzed using Affymetrix human transcriptomic array. Histological staining assessed structure, localization, and protein expression. Migration/proliferation was assessed using ORIS migration assay. We show different EMT types associated with BAV and TAV AscAA. Specifically, in BAV-associated aortopathy, EMT genes related to endocardial cushion formation were enriched. Further, BAV vascular smooth muscle cells were less proliferative and migratory. In contrast, TAV aneurysmal aortas displayed a fibrotic EMT phenotype with medial degenerative insults. Further, non-dilated BAV aortas showed a lower miRNA-200c-associated endothelial basement membrane LAMC1 expression and lower CD31 expression, accompanied by increased endothelial permeability indicated by increased albumin infiltration. Embryonic EMT is a characteristic of BAV aortopathy, associated with endothelial instability and vascular permeability of the non-dilated aortic wall. KEY MESSAGES: Embryonic EMT is a feature of BAV-associated aortopathy. Endothelial integrity is compromised in BAV aortas prior to dilatation. Non-dilated BAV ascending aortas are more permeable than aortas of tricuspid aortic valve patients.

Identification of TNFα-mediated inflammation as potential pathological marker and therapeutic target for calcification progress of congenital bicuspid aortic valve.

BACKGROUD: Congenital bicuspid aortic valve (cBAV) develops calcification and stenotic obstruction early compared with degenerative tricuspid aortic valve (dTAV), which requires surgical intervention. Here we report a comparative study of patients with cBAV or dTAV to identify risk factors associated with the rapid development of calcified bicuspid valves. METHODS: A total of 69 aortic valves (24 dTAV and 45 cBAV) were collected at the time of surgical aortic valve replacement for comparative clinical characteristics. Ten samples were randomly selected from each group for histology, pathology, and inflammatory factors expression and comparison analyses. OM-induced calcification in porcine aortic valve interstitial cell cultures were prepared for illustrating the underlying molecular mechanisms about calcification progress of cBAV and dTAV. RESULTS: We found that cBAV patients have increased cases of aortic valve stenosis compared with dTAV patients. Histopathological examinations revealed increased collagens deposition, neovascularization and infiltrations by inflammatory cells, especially T-lymphocytes and macrophages. We identified that tumor necrosis factor α (TNFα) and its regulated inflammatory cytokines are upregulated in cBAV. Further in vitro study indicated that TNFα-NFκB and TNFα-GSK3ß pathway accelerate aortic valve interstitial cells calcification, while inhibition of TNFα significantly delays this process. CONCLUSION: The finding of intensified TNFα-mediated inflammation in the pathological cBAV advocates the inhibition of TNFα as a potential treatment for patients with cBAV by alleviating the progress of inflammation-induced valve damage and calcification.

Abnormal mechanical stress on bicuspid aortic valve induces valvular calcification and inhibits Notch1/NICD/Runx2 signal.

Background: Bicuspid aortic valve (BAV) is a congenital cardiac deformity, increasing the risk of developing calcific aortic valve disease (CAVD). The disturbance of hemodynamics can induce valvular calcification, but the mechanism has not been fully identified. Methods: We constructed a finite element model (FEM) of the aortic valve based on the computed tomography angiography (CTA) data from BAV patients and tricuspid aortic valve (TAV) individuals. We analyzed the hemodynamic properties based on our model and investigated the characteristics of mechanical stimuli on BAV. Further, we detected the expression of Notch, NICD and Runx2 in valve samples and identified the association between mechanical stress and the Notch1 signaling pathway. Results: Finite element analysis showed that at diastole phase, the equivalent stress on the root of BAV was significantly higher than that on the TAV leaflet. Correspondingly, the expression of Notch1 and NICH decreased and the expression of Runx2 elevated significantly on large BAV leaflet belly, which is associated with equivalent stress on leaflet. Our findings indicated that the root of BAV suffered higher mechanical stress due to the abnormal hemodynamic environment, and the disturbance of the Notch1/NICD/Runx2 signaling pathway caused by mechanical stimuli contributed to valvular calcification.

Variations in the poly-histidine repeat motif of HOXA1 contribute to bicuspid aortic valve in mouse and zebrafish.

Bicuspid aortic valve (BAV), the most common cardiovascular malformation occurs in 0.5-1.2% of the population. Although highly heritable, few causal mutations have been identified in BAV patients. Here, we report the targeted sequencing of HOXA1 in a cohort of BAV patients and the identification of rare indel variants in the homopolymeric histidine tract of HOXA1. In vitro analysis shows that disruption of this motif leads to a significant reduction in protein half-life and defective transcriptional activity of HOXA1. In zebrafish, targeting hoxa1a ortholog results in aortic valve defects. In vivo assays indicates that these variants behave as dominant negatives leading abnormal valve development. In mice, deletion of Hoxa1 leads to BAV with a very small, rudimentary non-coronary leaflet. We also show that 17% of homozygous Hoxa1-1His knock-in mice present similar phenotype. Genetic lineage tracing in Hoxa1-/- mutant mice reveals an abnormal reduction of neural crest-derived cells in the valve leaflet, which is caused by a failure of early migration of these cells.

Noncanonical atherosclerosis as the driving force in tricuspid aortic valve associated aneurysms - A trace collection.

Pathogenic mechanisms in degenerative thoracic aortic aneurysms (TAA) are still unclear. There is an ongoing debate about whether TAAs are caused by uniform or distinct processes, which would obviously have a major impact on future treatment strategies. Clearly, the ultimate outcome of TAA subgroups associated with a tricuspid aortic valve (TAV) or a bicuspid aortic valve (BAV) is the same, namely a TAA. Based on results from our own and others' studies, we decided to compare the different TAAs (TAV and BAV) and controls using a broad array of analyses, i.e., metabolomic analyses, gene expression profiling, protein expression analyses, histological characterization, and matrix-assisted laser desorption ionization imaging. Central findings of the present study are the presence of noncanonical atherosclerosis, pathological accumulation of macrophages, and disturbances of lipid metabolism in the aortic media. Moreover, we have also found that lipid metabolism is impaired systemically. Importantly, all of the above-described phenotypes are characteristic for TAV-TAA only, and not for BAV-TAA. In summary, our results suggest different modes of pathogenesis in TAV- and BAV-associated aneurysms. Intimal atherosclerotic changes play a more central role in TAV-TAA formation than previously thought, particularly as the observed alterations do not follow classical patterns. Atherosclerotic alterations are not limited to the intima but also affect and alter the TAV-TAA media. Further studies are needed to i) clarify patho-relevant intima-media interconnections, ii) define the origin of the systemic alteration of lipid metabolism, and iii) to define valid biomarkers for early diagnosis, disease progression, and successful treatments in TAV-TAAs.

Genetics of aortic valve disease.

PURPOSE OF REVIEW: Aortic valve disease is a leading global cause of morbidity and mortality, posing an increasing burden on society. Advances in next-generation technologies and disease models over the last decade have further delineated the genetic and molecular factors that might be exploited in development of therapeutics for affected patients. This review describes several advances in the molecular and genetic understanding of AVD, focusing on bicuspid aortic valve (BAV) and calcific aortic valve disease (CAVD). RECENT FINDINGS: Genomic studies have identified a myriad of genes implicated in the development of BAV, including NOTCH1 , SMAD6 and ADAMTS19 , along with members of the GATA and ROBO gene families. Similarly, several genes associated with the initiation and progression of CAVD, including NOTCH1 , LPA , PALMD , IL6 and FADS1/2 , serve as the launching point for emerging clinical trials. SUMMARY: These new insights into the genetic contributors of AVD have offered new avenues for translational disease investigation, bridging molecular discoveries to emergent pharmacotherapeutic options. Future studies aimed at uncovering new genetic associations and further defining implicated molecular pathways are fuelling the new wave of drug discovery.

Elucidation of the genetic causes of bicuspid aortic valve disease.

AIMS: The present study aims to characterize the genetic risk architecture of bicuspid aortic valve (BAV) disease, the most common congenital heart defect. METHODS AND RESULTS: We carried out a genome-wide association study (GWAS) including 2236 BAV patients and 11 604 controls. This led to the identification of a new risk locus for BAV on chromosome 3q29. The single nucleotide polymorphism rs2550262 was genome-wide significant BAV associated (P = 3.49 × 10-08) and was replicated in an independent case-control sample. The risk locus encodes a deleterious missense variant in MUC4 (p.Ala4821Ser), a gene that is involved in epithelial-to-mesenchymal transformation. Mechanistical studies in zebrafish revealed that loss of Muc4 led to a delay in cardiac valvular development suggesting that loss of MUC4 may also play a role in aortic valve malformation. The GWAS also confirmed previously reported BAV risk loci at PALMD (P = 3.97 × 10-16), GATA4 (P = 1.61 × 10-09), and TEX41 (P = 7.68 × 10-04). In addition, the genetic BAV architecture was examined beyond the single-marker level revealing that a substantial fraction of BAV heritability is polygenic and ∼20% of the observed heritability can be explained by our GWAS data. Furthermore, we used the largest human single-cell atlas for foetal gene expression and show that the transcriptome profile in endothelial cells is a major source contributing to BAV pathology. CONCLUSION: Our study provides a deeper understanding of the genetic risk architecture of BAV formation on the single marker and polygenic level.

Structural dysregulation of the pulmonary autograft was associated with a greater density of p16INK4A-vascular smooth muscle cells.

The present study tested the hypothesis that a senescent phenotype of vascular smooth muscle cells (VSMCs) may represent the seminal event linked to maladaptive pulmonary autograft remodeling of a small number of patients that underwent the Ross procedure. The diameter of the pulmonary autograft (47±4 mm) of three male patients was significantly greater compared to the pulmonary artery (26±1 mm) excised from bicuspid aortic valve (BAV) patients. The pulmonary autograft was associated with a neointimal region and the adjacent medial region was significantly thinner compared to the pulmonary artery of BAV patients. Structural dysregulation was evident as elastin content of the medial region was significantly reduced in the pulmonary autograft compared to the pulmonary artery of BAV patients. By contrast, collagen content of the medial region of the pulmonary autograft and the pulmonary artery of BAV patients was not significantly different. Reduced medial elastin content of the pulmonary autograft was associated with increased protein levels of matrix metalloproteinase-9. The latter phenotype was not attributed to a robust inflammatory response as the percentage of Mac-2(+)-infiltrating monocytes/macrophages was similar between groups. A senescent phenotype was identified as protein levels of the cell cycle inhibitor p27kip1 were upregulated and the density of p16INK4A/non-muscle myosin IIB(+)-VSMCs was significantly greater in the pulmonary autograft compared to the pulmonary artery of BAV patients. Thus, senescent VSMCs may represent the predominant cellular source of increased matrix metalloproteinase-9 protein expression translating to maladaptive pulmonary autograft remodeling characterized by elastin degradation, medial thinning and neointimal formation.

Bicuspid Aortic Valve-Associated Regulatory Regions Reveal GATA4 Regulation and Function During Human-Induced Pluripotent Stem Cell-Based Endothelial-Mesenchymal Transition-Brief Report.

BACKGROUND: The endothelial-mesenchymal transition (EndoMT) is a fundamental process for heart valve formation and defects in EndoMT cause aortic valve abnormalities. Our previous genome-wide association study identified multiple variants in a large chromosome 8 segment as significantly associated with bicuspid aortic valve (BAV). The objective of this study is to determine the biological effects of this large noncoding segment in human induced pluripotent stem cell (hiPSC)-based EndoMT. METHODS: A large genomic segment enriched for BAV-associated variants was deleted in hiPSCs using 2-step CRISPR/Cas9 editing. To address the effects of the variants on GATA4 expression, we generated CRISPR repression hiPSC lines (CRISPRi) as well as hiPSCs from BAV patients. The resulting hiPSCs were differentiated to mesenchymal/myofibroblast-like cells through cardiovascular-lineage endothelial cells for molecular and cellular analysis. Single-cell RNA sequencing was also performed at different stages of EndoMT induction. RESULTS: The large deletion impaired hiPSC-based EndoMT in multiple biallelic clones compared with their isogenic control. It also reduced GATA4 transcript and protein levels during EndoMT, sparing the other genes nearby the deletion segment. Single-cell trajectory analysis revealed the molecular reprogramming during EndoMT. Putative GATA-binding protein targets during EndoMT were uncovered, including genes implicated in endocardial cushion formation and EndoMT process. Differentiation of cells derived from BAV patients carrying the rs117430032 variant as well as CRISPRi repression of the rs117430032 locus resulted in lower GATA4 expression in a stage-specific manner. TWIST1 was identified as a potential regulator of GATA4 expression, showing specificity to the locus tagged by rs117430032. CONCLUSIONS: BAV-associated distal regions regulate GATA4 expression during hiPSC-based EndoMT, which in turn promotes EndoMT progression, implicating its contribution to heart valve development.

Ciprofloxacin exacerbates dysfunction of smooth muscle cells in a microphysiological model of thoracic aortic aneurysm.

Ciprofloxacin use may be associated with adverse aortic events. However, the mechanism underlying the effect of ciprofloxacin on the progression of thoracic aortic aneurysm (TAA) is not well understood. Using an in vitro microphysiological model, we treated human aortic smooth muscle cells (HASMCs) derived from patients with bicuspid aortic valve- or tricuspid aortic valve-associated (BAV- or TAV-associated) TAAs with ciprofloxacin. TAA C57BL/6 mouse models were utilized to verify the effects of ciprofloxacin exposure. In the microphysiological model, real-time PCR, Western blotting, and RNA sequencing showed that ciprofloxacin exposure was associated with a downregulated contractile phenotype, an upregulated inflammatory reaction, and extracellular matrix (ECM) degradation in the normal HASMCs derived from the nondiseased aorta. Ciprofloxacin induced mitochondrial dysfunction in the HASMCs and further increased apoptosis by activating the ERK1/2 and P38 mitogen-activated protein kinase pathways. These adverse effects appeared to be more severe in the HASMCs derived from BAV- and TAV-associated TAAs than in the normal HASMCs when the ciprofloxacin concentration exceeded 100 µg/mL. In the aortic walls of the TAA-induced mice, ECM degradation and apoptosis were aggravated after ciprofloxacin exposure. Therefore, ciprofloxacin should be used with caution in patients with BAV- or TAV-associated TAAs.

Can transforming growth factor beta and downstream signalers distinguish bicuspid aortic valve patients susceptible for future aortic complications?

Patients with a bicuspid aortic valve have an extreme high risk to develop a thoracic aortic aneurysm and dissection (TAAD). TAADs form a leading cause of death worldwide, with the majority of deaths being preventable if individuals at risk are identified and properly managed. Risk stratification for TAADs in bicuspidy is so far solely based on the aortic diameter. Exclusive use of aortic wall dimension, as in the current guidelines, is however not sufficient in selecting patients vulnerable for future aortic wall complications. Moreover, there are no effective medical treatments for TAADs to retain progressive aortic dilatation and thus prevent or delay aortic complications. Only surgical replacement of the aorta increases life expectancy in patients with a risk for a TAAD. Therefore, the next major challenge in the management of TAADs is the development of a personalized patient-tailored risk stratification for early detection of patients with an increased risk for TAADs, who will benefit from surgical resection of the aorta. Several signaling pathways have been studied in recent times to develop a patient specific risk stratification model. In this paper we discuss TGF-ß signaling and downstream signalers as potential markers for future aortic complications in bicuspid aortic valve patients.

A Segmental Approach from Molecular Profiling to Medical Imaging to Study Bicuspid Aortic Valve Aortopathy.

Bicuspid aortic valve (BAV) patients develop ascending aortic (AAo) dilation. The pathogenesis of BAV aortopathy (genetic vs. haemodynamic) remains unclear. This study aims to identify regional changes around the AAo wall in BAV patients with aortopathy, integrating molecular data and clinical imaging. BAV patients with aortopathy (n = 15) were prospectively recruited to surgically collect aortic tissue and measure molecular markers across the AAo circumference. Dilated (anterior/right) vs. non-dilated (posterior/left) circumferential segments were profiled for whole-genomic microRNAs (next-generation RNA sequencing, miRCURY LNA PCR), protein content (tandem mass spectrometry), and elastin fragmentation and degeneration (histomorphometric analysis). Integrated bioinformatic analyses of RNA sequencing and proteomic datasets identified five microRNAs (miR-128-3p, miR-210-3p, miR-150-5p, miR-199b-5p, and miR-21-5p) differentially expressed across the AAo circumference. Among them, three miRNAs (miR-128-3p, miR-150-5p, and miR-199b-5p) were predicted to have an effect on eight common target genes, whose expression was dysregulated, according to proteomic analyses, and involved in the vascular-endothelial growth-factor signalling, Hippo signalling, and arachidonic acid pathways. Decreased elastic fibre levels and elastic layer thickness were observed in the dilated segments. Additionally, in a subset of patients n = 6/15, a four-dimensional cardiac magnetic resonance (CMR) scan was performed. Interestingly, an increase in wall shear stress (WSS) was observed at the anterior/right wall segments, concomitantly with the differentially expressed miRNAs and decreased elastic fibres. This study identified new miRNAs involved in the BAV aortic wall and revealed the concomitant expressional dysregulation of miRNAs, proteins, and elastic fibres on the anterior/right wall in dilated BAV patients, corresponding to regions of elevated WSS.

Transcription factor Sp2 promotes TGFB-mediated interstitial cell osteogenic differentiation in bicuspid aortic valves through a SMAD-dependent pathway.

Calcification of the bicuspid aortic valve (BAV) involves differential expression of various RNA genes, which is achieved through complex regulatory networks that are controlled in part by transcription factors and microRNAs. We previously found that miR-195-5p regulates the osteogenic differentiation of valvular interstitial cells (VICs) by targeting the TGF-ß pathway. However, the transcriptional regulation of miR-195-5p in calcified BAV patients is not yet clear. In this study, stenotic aortic valve tissues from patients with BAVs and tricuspid aortic valves (TAVs) were collected. Candidate transcription factors of miR-195-5p were predicted by bioinformatics analysis and tested in diseased valves and in male porcine VICs. SP2 gene expression and the corresponding protein levels in BAV were significantly lower than those in TAV, and a low SP2 expression level environment in VICs resulted in remarkable increases in RNA expression levels of RUNX2, BMP2, collagen 1, MMP2, and MMP9 and the corresponding proteins. ChIP assays revealed that SP2 directly bound to the transcription promoter region of miR-195-5p. Cotransfection of SP2 shRNA and a miR-195-5p mimic in porcine VICs demonstrated that SP2 repressed SMAD7 expression via miR-195-5p, while knockdown of SP2 increased the mRNA expression of SMAD7 and the corresponding protein and attenuated Smad 2/3 expression. Immunofluorescence staining of diseased valves confirmed that the functional proteins of osteogenesis differentiation, including RUNX2, BMP2, collagen 1, and osteocalcin, were overexpressed in BAVs. In Conclusion, the transcription factor Sp2 is expressed at low levels in VICs from BAV patients, which has a negative impact on miR-195-5p expression by binding its promoter region and partially promotes calcification through a SMAD-dependent pathway.

Medial tunica degeneration of the ascending aortic wall is associated with specific microRNA changes in bicuspid aortic valve disease.

Ascending aortic diameter is not an accurate parameter for surgical indication in patients with bicuspid aortic valve (BAV). Thus, the present study aimed to identify specific microRNAs (miRNAs/miRs) and their expression levels in aortic wall aneurysm associated with BAV according to severity of medial degeneration and to elucidate the association between the tissue expression levels of the miRNAs with their expression in plasma. Aortic wall and blood specimens were obtained from 38 patients: 12 controls and 26 patients with BAV with ascending aortic aneurysm. Of the patients with BAV, 19 had cusp fusions of right and left, 5 of right and non­coronary, and 2 of left and non­coronary. Two groups of patients were identified according to the grade of medial degeneration (MD): Low­grade D group (LGMD) and high­grade MD group (HGMD). Expression level of miR­122, miR­130, miR­718 and miR­486 were validated by reverse transcription­quantitative PCR in plasma and tissue samples. MD grade was found to be independent from the BAV phenotype. The HGD group showed increased expression levels of MMP­9 and MMP­2, and an increase in the number of apoptotic cells. Tissue expression levels of miR­718 and miR­122 were lower in the LGMD and HGD groups compared with expression in the control group; the HGD group showed increased levels of miR­486. Plasma expression levels of miR­122 were decreased in the LGMD and HGD groups, and miR­718 was only reduced in the HGD group. On the contrary, expression of miR­486 was increased in the LGMD and HGD groups. The data suggested that miR­486 may be considered as a non­invasive biomarker of aortic wall degeneration. Dysregulation of this putative biomarker may be associated with high risk of dissection and rupture in patients with BAV.

Aorta smooth muscle-on-a-chip reveals impaired mitochondrial dynamics as a therapeutic target for aortic aneurysm in bicuspid aortic valve disease.

Background: Bicuspid aortic valve (BAV) is the most common congenital cardiovascular disease in general population and is frequently associated with the development of thoracic aortic aneurysm (TAA). There is no effective strategy to intervene with TAA progression due to an incomplete understanding of the pathogenesis. Insufficiency of NOTCH1 expression is highly related to BAV-TAA, but the underlying mechanism remains to be clarified. Methods: A comparative proteomics analysis was used to explore the biological differences between non-diseased and BAV-TAA aortic tissues. A microfluidics-based aorta smooth muscle-on-a-chip model was constructed to evaluate the effect of NOTCH1 deficiency on contractile phenotype and mitochondrial dynamics of human aortic smooth muscle cells (HAoSMCs). Results: Protein analyses of human aortic tissues showed the insufficient expression of NOTCH1 and impaired mitochondrial dynamics in BAV-TAA. HAoSMCs with NOTCH1-knockdown exhibited reduced contractile phenotype and were accompanied by attenuated mitochondrial fusion. Furthermore, we identified that mitochondrial fusion activators (leflunomide and teriflunomide) or mitochondrial fission inhibitor (Mdivi-1) partially rescued the disorders of mitochondrial dynamics in HAoSMCs derived from BAV-TAA patients. Conclusions: The aorta smooth muscle-on-a-chip model simulates the human pathophysiological parameters of aorta biomechanics and provides a platform for molecular mechanism studies of aortic disease and related drug screening. This aorta smooth muscle-on-a-chip model and human tissue proteomic analysis revealed that impaired mitochondrial dynamics could be a potential therapeutic target for BAV-TAA. Funding: National Key R and D Program of China, National Natural Science Foundation of China, Shanghai Municipal Science and Technology Major Project, Shanghai Science and Technology Commission, and Shanghai Municipal Education Commission.

To function properly, the heart must remain a one-way system, pumping out oxygenated blood into the aorta ­ the largest artery in the body ­ so it can be distributed across the organism. The aortic valve, which sits at the entrance of the aorta, is a key component of this system. Its three flaps (or 'cusps') are pushed open when the blood exits the heart, and they shut tightly so it does not flow back in the incorrect direction. Nearly 1.4% of people around the world are born with 'bicuspid' aortic valves that only have two flaps. These valves may harden or become leaky, forcing the heart to work harder. This defect is also associated with bulges on the aorta which progressively weaken the artery, sometimes causing it to rupture. Open-heart surgery is currently the only way to treat these bulges (or 'aneurysms'), as no drug exists that could slow down disease progression. This is partly because the biological processes involved in the aneurysms worsening and bursting open is unclear. Recent studies have highlighted that many individuals with bicuspid aortic valves also have lower levels of a protein known as NOTCH1, which plays a key signalling role for cells. Problems in the mitochondria ­ the structures that power up a cell ­ are also observed. However, it is not known how these findings are connected or linked with the aneurysms developing. To answer this question, Abudupataer et al. analyzed the proteins present in diseased and healthy aortic muscle cells, confirming a lower production of NOTCH1 and impaired mitochondria in diseased tissues. They also created an 'aorta-on-a-chip' model where aortic muscle cells were grown in the laboratory under conditions resembling those found in the body ­ including the rhythmic strain that the aorta is under because of the heart beating. Abudupataer et al. then reduced NOTCH1 levels in healthy samples, which made the muscle tissue less able to contract and reduced the activity of the mitochondria. Applying drugs that tweak mitochondrial activity helped tissues from patients with bicuspid aortic valves to work better. These compounds could potentially benefit individuals with deficient aortic valves, but experiments in animals and clinical trials would be needed first to confirm the results and assess safety. The aorta-on-a-chip model developed by Abudupataer et al. also provides a platform to screen for drugs and examine the molecular mechanisms at play in aortic diseases.

Urinary Metabolomics Study of Patients with Bicuspid Aortic Valve Disease.

Bicuspid aortic valve (BAV) is the most common congenital heart defect responsible for valvular and aortic complications in affected patients. Causes and mechanisms of this pathology are still elusive and thus the lack of early detection biomarkers leads to challenges in its diagnosis and prevention of associated cardiovascular anomalies. The aim of this study was to explore the potential use of urine Nuclear Magnetic Resonance (NMR) metabolomics to evaluate a molecular fingerprint of BAV. Both multivariate and univariate statistical analyses were performed to compare the urinary metabolome of 20 patients with BAV with that of 24 matched controls. Orthogonal partial least squared discriminant analysis (OPLS-DA) showed statistically significant discrimination between cases and controls, suggesting seven metabolites (3-hydroxybutyrate, alanine, betaine, creatine, glycine, hippurate, and taurine) as potential biomarkers. Among these, glycine, hippurate and taurine individually displayed medium sensitivity and specificity by receiver operating characteristic (ROC) analysis. Pathway analysis indicated two metabolic pathways likely perturbed in BAV subjects. Possible contributions of gut microbiota activity and energy imbalance are also discussed. These results constitute encouraging preliminary findings in favor of the use of urine-based metabolomics for early diagnosis of BAV.

Recurrent germline mutations as genetic markers for aortic root dilatation in bicuspid aortic valve patients.

Bicuspid aortic valve (BAV) is characterized by elevated risk of aortic dilatation and aneurysm. Although genetic susceptibility is suspected to influence on the development of BAV aortopathy, clinical application of genetic markers still needs validation in BAV entities with strictly defined phenotypic features. The 'root phenotype' represents a young, male predominant, and severely aortic regurgitant BAV population prone to aortic root dilatation. The present study launched a two-step genetic survey to evaluate the clinical significance of germline genetic markers in BAV patients. The whole-exome sequencing (WES) cohort consisted of 13 BAV patients with 'root phenotype' under the age of 40 years. We identified 28 different heterozygous missense mutations in 19 genes from the WES cohort, among which six variants (COL1A2 R882C, COL5A1 I1161F, ACVRL1 R218W, NOTCH1 P1227S, MYLK S243W, MYLK D717Y) were identified as pathogenic variants via unanimous agreement of in silico prediction tool analysis, and three variants (C1R I345L, TGFBR2 V216I, FBN2 G475V) were identified as recurrent variants. The panel of nine genetic markers was tested in an independent validation cohort of 154 BAV patients consecutively included from January to May 2018 in our institution. The validation cohort demonstrated 71.4% male predominance and the average age of 57 ± 13 years, among which 26.6% showed aortic root dilatation and 66.9% ascending aortic dilatation. Genetic markers were found in 32 patients, including 18 with C1R I345L, 11 with TGFBR2 V216I, 2 with FBN2 G475V, and 1 with both TGFBR2 V216I and MYLK D717Y. BAV patients carrying these genetic markers demonstrated younger age [(51 ± 12) vs. (58 ± 13) years, P = 0.014], more moderate to severe aortic regurgitation (56.2% vs. 33.6%, P = 0.019), elevated prevalence of mitral valve prolapse (9.4% vs. 0.8%, P = 0.028) and aortic root dilatation (62.5% vs. 17.2%, P < 0.001) but not ascending aortic dilatation than those without these markers. The early-onset 'root phenotype' entities displayed great value for BAV genetic surveys. As one of the promising complements of the current risk stratification system, recurrent germline mutations in TGFBR2, C1R, FBN2 genes could be identified and applied as genetic markers of elevated susceptibility for aortic root but not ascending aortic dilatation among BAV patients.