Oxygenator assisted dynamic microphysiological culture elucidates the impact of hypoxia on valvular interstitial cell calcification.

INTRODUCTION: Microphysiological systems (MPS) offer simulation of (patho)physiological parameters. Investigation includes items which lead to fibrosis and calcification in development and progress of calcific aortic valve disease, based e.g. on culturing of isolated valvular interstitial cells (VICs). Hypoxia regulated by hypoxia inducible factors impacts pathological differentiation in aortic valve (AV) disease. This is mimicked via an MPS implemented oxygenator in combination with calcification inducing medium supplementation. METHODS: Human valvular interstitial cells were isolated and dynamically cultured in MPS at hypoxic, normoxic, arterial blood oxygen concentration and cell incubator condition. Expression profile of fibrosis and calcification markers was monitored and calcification was quantified in induction and control media with and without hypoxia and in comparison to statically cultured counterparts. RESULTS: Hypoxic 24-hour culture of human VICs leads to HIF1α nuclear localization and induction of EGLN1, EGLN3 and LDHA mRNA expression but does not directly impact expression of fibrosis and calcification markers. Dependent on medium formulation, induction medium induces monolayer calcification and elevates RUNX2, ACTA2 and FN1 but reduces SOX9 mRNA expression in dynamic and static MPS culture. But combining hypoxic oxygen concentration leads to higher calcification potential of human VICs in calcification and standard medium formulation dynamically cultured for 96 h. CONCLUSION: In hypoxic oxygen concentration an increased human VIC calcification in 2D VIC culture in an oxygenator assisted MPS was detected. Oxygen regulation therefore can be combined with calcification induction media to monitor additional effects of pathological marker expression. Validation of oxygenator dependent VIC behavior envisions future advancement and transfer to long term aortic valve tissue culture MPS.

Observational and genetic association of non-alcoholic fatty liver disease and calcific aortic valve disease.

Background: Non-alcoholic fatty liver disease (NAFLD) has emerged as a predominant driver of chronic liver disease globally and is associated with increased cardiovascular disease morbidity and mortality. However, the association between NAFLD and calcific aortic valve disease remains unclear. We aimed to prospectively investigate the association between NAFLD and incident aortic valve calcification (AVC), as well as its genetic relationship with incident calcific aortic valve stenosis (CAVS). Methods: A post hoc analysis was conducted on 4226 participants from the Multi-Ethnic Study of Atherosclerosis (MESA) database. We employed the adjusted Cox models to assess the observational association between NAFLD and incident AVC. Additionally, we conducted two-sample Mendelian randomization (MR) analyses to investigate the genetic association between genetically predicted NAFLD and calcific aortic valve stenosis (CAVS), a severe form of CAVD. We repeated the MR analyses by excluding NAFLD susceptibility genes linked to impaired very low-density lipoprotein (VLDL) secretion. Results: After adjustment for potential risk factors, participants with NAFLD had a hazard ratio of 1.58 (95% CI: 1.03-2.43) for incident AVC compared to those without NAFLD. After excluding genes associated with impaired VLDL secretion, the MR analyses consistently showed the significant associations between genetically predicted NAFLD and CAVS for 3 traits: chronic elevation of alanine aminotransferase (odds ratio = 1.13 [95% CI: 1.01-1.25]), imaging-based NAFLD (odds ratio = 2.81 [95% CI: 1.66-4.76]), and biopsy-confirmed NAFLD (odds ratio = 1.12 [95% CI: 1.01-1.24]). However, the association became non-significant when considering all NAFLD susceptibility genes. Conclusions: NAFLD was independently associated with an elevated risk of incident AVC. Genetically predicted NAFLD was also associated with CAVS after excluding genetic variants related to impaired VLDL secretion.

Molecular Interaction of Soluble Klotho with FGF23 in the Pathobiology of Aortic Valve Lesions Induced by Chronic Kidney Disease.

Chronic kidney disease (CKD) is linked to greater prevalence and rapid progression of calcific aortic valve disease (CAVD) characterized by valvular leaflet fibrosis and calcification. Fibroblast growth factor 23 (FGF23) level is elevated, and anti-aging protein Klotho is reduced in CKD patients. However, the roles of FGF23 and Klotho in the mechanism of aortic valve fibrosis and calcification remain unclear. We hypothesized that FGF23 mediates CKD-induced CAVD by enhancing aortic valve interstitial cell (AVIC) fibrosis and calcification, while soluble Klotho inhibits FGF23 effect. Methods and Results: In an old mouse model of CKD, kidney damages were accompanied by aortic valve thickening and calcification. FGF23 levels in plasma and aortic valve were increased, while Klotho levels were decreased. Recombinant FGF23 elevated the inflammatory, fibrogenic, and osteogenic activities in AVICs. Neutralizing antibody or shRNA targeting FGF23 suppressed the pathobiological activities in AVICs from valves affected by CAVD. FGF23 exerts its effects on AVICs via FGF receptor (FGFR)/Yes-associated protein (YAP) signaling, and inhibition of FGFR/YAP reduced FGF23's potency in AVICs. Recombinant Klotho downregulated the pathobiological activities in AVICs exposed to FGF23. Incubation of FGF23 with Klotho formed complexes and decreased FGF23's potency. Further, treatment of CKD mice with recombinant Klotho attenuated aortic valve lesions. Conclusion: This study demonstrates that CKD induces FGF23 accumulation, Klotho insufficiency and aortic valve lesions in old mice. FGF23 upregulates the inflammatory, fibrogenic and osteogenic activities in AVICs via the FGFR/YAP signaling pathway. Soluble Klotho suppresses FGF23 effect through molecular interaction and is capable of mitigating CKD-induced CAVD.

Beyond VICs: Shedding light on the overlooked VECs in calcific aortic valve disease.

Calcific aortic valve disease (CAVD) is prevalent in developed nations and has emerged as a pressing global public health concern due to population aging. The precise etiology of this disease remains uncertain, and recent research has primarily focused on examining the role of valvular interstitial cells (VICs) in the development of CAVD. The predominant treatment options currently available involve open surgery and minimally invasive interventional surgery, with no efficacious pharmacological treatment. This article seeks to provide a comprehensive understanding of valvular endothelial cells (VECs) from the aspects of valvular endothelium-derived nitric oxide (NO), valvular endothelial mechanotransduction, valvular endothelial injury, valvular endothelial-mesenchymal transition (EndMT), and valvular neovascularization, which have received less attention, and aims to establish their role and interaction with VICs in CAVD. The ultimate goal is to provide new perspectives for the investigation of non-invasive treatment options for this disease.

TRPM4 mRNA stabilization by METTL3-mediated m6A modification promotes calcific aortic valve inflammation.

Background: Transient receptor potential melastatin 4 (TRPM4) affects immune responses by regulating calcium homeostasis, but its role in calcific aortic valve inflammation remains unclear. This study aimed to assess the expression and function of TRPM4 in patients with or without calcific aortic valve disease (CAVD). Methods: The mRNA and protein expression levels of TRPM4 and related factors in calcified and noncalcified tissues were measured using qRT-PCR and Western blot. The proteins interacting with TRPM4 were confirmed by RNA pull-down and RNA immunoprecipitation assays. Dual-Luciferase Reporter Assay was performed to confirm the m6A site of TRPM4. Results: The mRNA expression levels of TRPM4, TLR4, IL-6, MCP-1, TNF-α, and NF-κB p65 were significantly higher in calcified aortic valve tissues than in noncalcified tissues, and TRPM4 was significantly positively correlated with inflammation-related factors. The protein expression level of TRPM4, TLR4 and NF-κB p65 were significantly higher in calcified aortic valve tissues than in noncalcified tissues. N6-methyladenosine (m6A) modification of TRPM4 mRNA by METTL3-YTHDF1 up-regulated its expression in CAVD. And TRPM4 promoted the level of inflammation via activation of the JNK-MAPK signaling pathway, after knockdown TRPM4, the production of proinflammatory cytokines was significantly suppressed. Conclusion: The results indicate the pivotal role of TRPM4 in CAVD and highlight METTL3-mediated m6A modification of TRPM4 in promoting inflammation through JNK-MAPK signaling pathway. This work provides potential therapeutic strategy to impede inflammation in CAVD.

Nesfatin-1 mitigates calcific aortic valve disease via suppressing ferroptosis mediated by GSH/GPX4 and ZIP8/SOD2 axes.

OBJECTIVE: Calcific aortic valve disease (CAVD) predominantly affects the elderly and currently lacks effective medical treatments. Nesfatin-1, a peptide derived from the cleavage of Nucleobindin 2, has been implicated in various calcification processes, both physiological and pathological. This study explores the impact of Nesfatin-1 on the transformation of aortic valve interstitial cells (AVICs) in CAVD. METHODS AND RESULTS: In vitro experiments showed that Nesfatin-1 treatment mitigated the osteogenic differentiation of AVICs. Corresponding in vivo studies demonstrated a deceleration in the progression of CAVD. RNA-sequencing of AVICs treated with and without Nesfatin-1 highlighted an enrichment of the Ferroptosis pathway among the top pathways identified by the Kyoto Encyclopedia of Genes and Genomes analysis. Further examination confirmed increased ferroptosis in both calcified valves and osteoblast-like AVICs, with a reduction in ferroptosis following Nesfatin-1 treatment. Within the Ferroptosis pathway, ZIP8 showed the most notable modulation by Nesfatin-1. Silencing ZIP8 in AVICs increased ferroptosis and osteogenic differentiation, decreased intracellular Mn2+ concentration, and reduced the expression and activity of superoxide dismutase (SOD2). Furthermore, the silencing of SOD2 exacerbated ferroptosis and osteogenic differentiation. Nesfatin-1 treatment was found to elevate the expression of glutathione peroxidase 4 (GPX4) and levels of glutathione (GSH), as confirmed by Western blotting and GSH concentration assays. CONCLUSION: In summary, Nesfatin-1 effectively inhibits the osteogenic differentiation of AVICs by attenuating ferroptosis, primarily through the GSH/GPX4 and ZIP8/SOD2 pathways.

FOXO1 regulates RUNX2 ubiquitination through SMURF2 in calcific aortic valve disease.

The prevalence of calcific aortic valve disease (CAVD) remains substantial while there is currently no medical therapy available. Forkhead box O1 (FOXO1) is known to be involved in the pathogenesis of cardiovascular diseases, including vascular calcification and atherosclerosis; however, its specific role in calcific aortic valve disease remains to be elucidated. In this study, we identified FOXO1 significantly down-regulated in the aortic valve interstitial cells (VICs) of calcified aortic valves by investigating clinical specimens and GEO database analysis. FOXO1 silencing or inhibition promoted VICs osteogenic differentiation in vitro and aortic valve calcification in Apoe-/- mice, respectively. We identified that FOXO1 facilitated the ubiquitination and degradation of RUNX2, which process was mainly mediated by SMAD-specific E3 ubiquitin ligase 2 (SMURF2). Our discoveries unveil a heretofore unacknowledged mechanism involving the FOXO1/SMURF2/RUNX2 axis in CAVD, thereby proposing the potential therapeutic utility of FOXO1 or SMURF2 as viable strategies to impede the progression of CAVD.

Investigation of autophagy­related genes and immune infiltration in calcific aortic valve disease: A bioinformatics analysis and experimental validation.

The present study aimed to elucidate the role of autophagy-related genes (ARGs) in calcific aortic valve disease (CAVD) and their potential interactions with immune infiltration via experimental verification and bioinformatics analysis. A total of three microarray datasets (GSE12644, GSE51472 and GSE77287) were obtained from the Gene Expression Omnibus database, and gene set enrichment analysis was performed to identify the relationship between autophagy and CAVD. After differentially expressed genes and differentially expressed ARGs (DEARGs) were identified using CAVD samples and normal aortic valve samples, a functional analysis was performed, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses, protein-protein interaction network construction, hub gene identification and validation, immune infiltration and drug prediction. The results of the present study indicated a significant relationship between autophagy and CAVD. A total of 46 DEARGs were identified. GO and pathway enrichment analyses revealed the complex roles of DEARGs in regulating CAVD, including multiple gene functions and pathways. A total of 10 hub genes were identified, with three (SPP1, CXCL12 and CXCR4) consistently upregulated in CAVD samples compared with normal aortic valve samples in multiple datasets and experimental validation. Immune infiltration analyses demonstrated significant differences in immune cell proportions between CAVD samples and normal aortic valve samples, thus showing the crucial role of immune infiltration in CAVD development. Furthermore, therapeutic drugs were predicted that could target the identified hub genes, including bisphenol A, resveratrol, progesterone and estradiol. In summary, the present study illuminated the crucial role of autophagy in CAVD development and identified key ARGs as potential therapeutic targets. In addition, the observed immune cell infiltration and predicted autophagy-related drugs suggest promising avenues for future research and novel CAVD treatments.

Morusin Alleviates Aortic Valve Calcification by Inhibiting Valve Interstitial Cell Senescence Through Ccnd1/Trim25/Nrf2 Axis.

The senescence of aortic valve interstitial cells (VICs) plays a critical role in the progression of calcific aortic valve disease (CAVD). However, the precise mechanisms underlying the senescence of VICs remain unclear, demanding the identification of a novel target to mitigate this process. Previous studies have highlighted the anti-aging potential of morusin. Thus, this study aimed to explore the therapeutic potential of morusin in CAVD. Cellular experiments reveal that morusin effectively suppresses cellular senescence and cause a shift toward osteogenic differentiation of VICs in vitro. Mechanistically, morusin activate the Nrf2-mediated antiaging signaling pathway by downregulating CCND1 expression and aiding Keap1 degradation through Trim 25. This activation lead to the upregulated expression of antioxidant genes, thus reducing reactive oxygen species production and thereby preventing VIC osteogenic differentiation. In vivo experiments in ApoE-/- mice on a high-fat Western diet demonstrate the positive effect of morusin in mitigating aortic valve calcification. These findings emphasize the antiaging properties of morusin and its potential as a therapeutic agent for CAVD.

Autotaxin inhibition attenuates the aortic valve calcification by suppressing inflammation-driven fibro-calcific remodeling of valvular interstitial cells.

BACKGROUND: Patients with fibro-calcific aortic valve disease (FCAVD) have lipid depositions in their aortic valve that engender a proinflammatory impetus toward fibrosis and calcification and ultimately valve leaflet stenosis. Although the lipoprotein(a)-autotaxin (ATX)-lysophosphatidic acid axis has been suggested as a potential therapeutic target to prevent the development of FCAVD, supportive evidence using ATX inhibitors is lacking. We here evaluated the therapeutic potency of an ATX inhibitor to attenuate valvular calcification in the FCAVD animal models. METHODS: ATX level and activity in healthy participants and patients with FCAVD were analyzed using a bioinformatics approach using the Gene Expression Omnibus datasets, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, and western blotting. To evaluate the efficacy of ATX inhibitor, interleukin-1 receptor antagonist-deficient (Il1rn-/-) mice and cholesterol-enriched diet-induced rabbits were used as the FCAVD models, and primary human valvular interstitial cells (VICs) from patients with calcification were employed. RESULTS: The global gene expression profiles of the aortic valve tissue of patients with severe FCAVD demonstrated that ATX gene expression was significantly upregulated and correlated with lipid retention (r = 0.96) or fibro-calcific remodeling-related genes (r = 0.77) in comparison to age-matched non-FCAVD controls. Orally available ATX inhibitor, BBT-877, markedly ameliorated the osteogenic differentiation and further mineralization of primary human VICs in vitro. Additionally, ATX inhibition significantly attenuated fibrosis-related factors' production, with a detectable reduction of osteogenesis-related factors, in human VICs. Mechanistically, ATX inhibitor prohibited fibrotic changes in human VICs via both canonical and non-canonical TGF-ß signaling, and subsequent induction of CTGF, a key factor in tissue fibrosis. In the in vivo FCAVD model system, ATX inhibitor exposure markedly reduced calcific lesion formation in interleukin-1 receptor antagonist-deficient mice (Il1rn-/-, P = 0.0210). This inhibition ameliorated the rate of change in the aortic valve area (P = 0.0287) and mean pressure gradient (P = 0.0249) in the FCAVD rabbit model. Moreover, transaortic maximal velocity (Vmax) was diminished with ATX inhibitor administration (mean Vmax = 1.082) compared to vehicle control (mean Vmax = 1.508, P = 0.0221). Importantly, ATX inhibitor administration suppressed the effects of a high-cholesterol diet and vitamin D2-driven fibrosis, in association with a reduction in macrophage infiltration and calcific deposition, in the aortic valves of this rabbit model. CONCLUSIONS: ATX inhibition attenuates the development of FCAVD while protecting against fibrosis and calcification in VICs, suggesting the potential of using ATX inhibitors to treat FCAVD.

Identification of hub genes in calcific aortic valve disease.

Calcific aortic valve disease (CAVD) is a heart valve disorder characterized primarily by calcification of the aortic valve, resulting in stiffness and dysfunction of the valve. CAVD is prevalent among aging populations and is linked to factors such as hypertension, dyslipidemia, tobacco use, and genetic predisposition, and can result in becoming a growing economic and health burden. Once aortic valve calcification occurs, it will inevitably progress to aortic stenosis. At present, there are no medications available that have demonstrated effectiveness in managing or delaying the progression of the disease. In this study, we mined four publicly available microarray datasets (GSE12644 GSE51472, GSE77287, GSE233819) associated with CAVD from the GEO database with the aim of identifying hub genes associated with the occurrence of CAVD and searching for possible biological targets for the early prevention and diagnosis of CAVD. This study provides preliminary evidence for therapeutic and preventive targets for CAVD and may provide a solid foundation for subsequent biological studies.

Relationship between peak aortic jet velocity and progression of aortic stenosis in patients undergoing hemodialysis.

BACKGROUND: The natural history of aortic stenosis (AS) progression, especially before severe AS development, is not well documented. We aimed to investigate the time course of peak aortic jet velocity (Vmax) and AS progression risk according to baseline Vmax, particularly whether there is a Vmax threshold. METHODS: In a retrospective multicenter cohort study of patients on hemodialysis with aortic valve calcification, we investigated the time series of Vmax and the relationship between the baseline Vmax and progression to severe AS by analyzing longitudinal echocardiographic data. RESULTS: Among 758 included patients (mean age, 71 years; 65% male), patients with Vmax <1.5, 1.5-1.9, 2.0-2.4, 2.5-2.9, and 3.0-3.9 m/s were 395 (52%), 216 (29%), 85 (11%), 39 (5.1%), and 23 (3.0%), respectively. The Vmax slope was gradual (mean 0.05-0.07 m/s/year) at Vmax <2 m/s, but steeper (mean 0.13-0.21 m/s/year) at Vmax ≥2 m/s. During a median 3.2-year follow-up, 52 (6.9%) patients developed severe AS. While patients with Vmax <2 m/s rarely developed severe AS, the risk of those with Vmax ≥2 m/s increased remarkably with an increasing baseline Vmax; the adjusted incidence rates in patients with Vmax <1.5, 1.5-1.9, 2.0-2.4, 2.5-2.9, and 3.0-3.9 m/s were 0.59, 0.57, 4.25, 13.8, and 56.1 per 100 person-years, respectively; the adjusted hazard ratio per 0.2 m/s increase in the baseline Vmax was 1.49 (95% confidence interval: 1.32-1.68) when Vmax ≥2 m/s. CONCLUSIONS: The risk of progression to severe AS increased with the baseline Vmax primarily at ≥2 m/s; a Vmax threshold of 2 m/s was observed.

Identification of pyroptosis-associated genes with diagnostic value in calcific aortic valve disease.

Background: Calcific aortic valve disease (CAVD) is one of the most prevalent valvular diseases and is the second most common cause for cardiac surgery. However, the mechanism of CAVD remains unclear. This study aimed to investigate the role of pyroptosis-related genes in CAVD by performing comprehensive bioinformatics analysis. Methods: Three microarray datasets (GSE51472, GSE12644 and GSE83453) and one RNA sequencing dataset (GSE153555) were obtained from the Gene Expression Omnibus (GEO) database. Pyroptosis-related differentially expressed genes (DEGs) were identified between the calcified and the normal valve samples. LASSO regression and random forest (RF) machine learning analyses were performed to identify pyroptosis-related DEGs with diagnostic value. A diagnostic model was constructed with the diagnostic candidate pyroptosis-related DEGs. Receiver operating characteristic (ROC) curve analysis was performed to estimate the diagnostic performances of the diagnostic model and the individual diagnostic candidate genes in the training and validation cohorts. CIBERSORT analysis was performed to estimate the differences in the infiltration of the immune cell types. Pearson correlation analysis was used to investigate associations between the diagnostic biomarkers and the immune cell types. Immunohistochemistry was used to validate protein concentration. Results: We identified 805 DEGs, including 319 down-regulated genes and 486 up-regulated genes. These DEGs were mainly enriched in pathways related to the inflammatory responses. Subsequently, we identified 17 pyroptosis-related DEGs by comparing the 805 DEGs with the 223 pyroptosis-related genes. LASSO regression and RF algorithm analyses identified three CAVD diagnostic candidate genes (TREM1, TNFRSF11B, and PGF), which were significantly upregulated in the CAVD tissue samples. A diagnostic model was constructed with these 3 diagnostic candidate genes. The diagnostic model and the 3 diagnostic candidate genes showed good diagnostic performances with AUC values >0.75 in both the training and the validation cohorts based on the ROC curve analyses. CIBERSORT analyses demonstrated positive correlation between the proportion of M0 macrophages in the valve tissues and the expression levels of TREM1, TNFRSF11B, and PGF. Conclusion: Three pyroptosis-related genes (TREM1, TNFRSF11B and PGF) were identified as diagnostic biomarkers for CAVD. These pyroptosis genes and the pro-inflammatory microenvironment in the calcified valve tissues are potential therapeutic targets for alleviating CAVD.

Transcriptomic Analysis of Mineralized Adipose-Derived Stem Cell Tissues for Calcific Valve Disease Modelling.

Calcific aortic valve disease (CAVD) is characterized by the fibrosis and mineralization of the aortic valve, which leads to aortic stenosis and heart failure. At the cellular level, this is due to the osteoblastic-like differentiation of valve interstitial cells (VICs), resulting in the calcification of the tissue. Unfortunately, human VICs are not readily available to study CAVD pathogenesis and the implicated mechanisms in vitro; however, adipose-derived stromal/stem cells (ASCs), carrying the patient's specific genomic features, have emerged as a promising cell source to model cardiovascular diseases due to their multipotent nature, availability, and patient-specific characteristics. In this study, we describe a comprehensive transcriptomic analysis of tissue-engineered, scaffold-free, ASC-embedded mineralized tissue sheets using bulk RNA sequencing. Bioinformatic and gene set enrichment analyses revealed the up-regulation of genes associated with the organization of the extracellular matrix (ECM), suggesting that the ECM could play a vital role in the enhanced mineralization observed in these tissue-engineered ASC-embedded sheets. Upon comparison with publicly available gene expression datasets from CAVD patients, striking similarities emerged regarding cardiovascular diseases and ECM functions, suggesting a potential link between ECM gene expression and CAVDs pathogenesis. A matrisome-related sub-analysis revealed the ECM microenvironment promotes the transcriptional activation of the master gene runt-related transcription factor 2 (RUNX2), which is essential in CAVD development. Tissue-engineered ASC-embedded sheets with enhanced mineralization could be a valuable tool for research and a promising avenue for the identification of more effective aortic valve replacement therapies.

Inhibition of valve mesenchymal stromal cell calcium deposition by bFGF through alternative polyadenylation regulation of the CAT gene.

OBJECTIVE: Calcific aortic valve disease (CAVD) is the leading cause of angina, heart failure, and death from aortic stenosis. However, the molecular mechanisms of its progression, especially the complex disease-related transcriptional regulatory mechanisms, remain to be further elucidated. METHODS: This study used porcine valvular interstitial cells (PVIC) as a model. We used osteogenic induced medium (OIM) to induce calcium deposition in PVICs to calcify them, followed by basic fibroblast growth factor (bFGF) treatment to inhibit calcium deposition. Transcriptome sequencing was used to study the mRNA expression profile of PVICs and its related transcriptional regulation. We used DaPars to further examine alternative polyadenylation (APA) between different treatment groups. RESULTS: We successfully induced calcium deposition of PVICs through OIM. Subsequently, mRNA-seq was used to identify differentially expressed mRNAs for three different treatments: control, OIM-induced and OIM-induced bFGF treatment. Global APA events were identified in the OIM and bFGF treatment groups by bioinformatics analysis. Finally, it was discovered and proven that catalase (CAT) is one of the potential targets of bFGF-induced APA regulation. CONCLUSION: We described a global APA change in a calcium deposition model related to CAVD. We revealed that transcriptional regulation of the CAT gene may contribute to bFGF-induced calcium deposition inhibition.

Models for calcific aortic valve disease in vivo and in vitro.

Calcific Aortic Valve Disease (CAVD) is prevalent among the elderly as the most common valvular heart disease. Currently, no pharmaceutical interventions can effectively reverse or prevent CAVD, making valve replacement the primary therapeutic recourse. Extensive research spanning decades has contributed to the establishment of animal and in vitro cell models, which facilitates a deeper understanding of the pathophysiological progression and underlying mechanisms of CAVD. In this review, we provide a comprehensive summary and analysis of the strengths and limitations associated with commonly employed models for the study of valve calcification. We specifically emphasize the advancements in three-dimensional culture technologies, which replicate the structural complexity of the valve. Furthermore, we delve into prospective recommendations for advancing in vivo and in vitro model studies of CAVD.

SIRT6-mediated Runx2 downregulation inhibits osteogenic differentiation of human aortic valve interstitial cells in calcific aortic valve disease.

Calcific aortic valve disease (CAVD) is a progressive cardiovascular disorder involving multiple pathogenesis. Effective pharmacological therapies are currently unavailable. Sirtuin6 (SIRT6) has been shown to protect against aortic valve calcification in CAVD. The exact regulatory mechanism of SIRT6 in osteoblastic differentiation remains to be determined, although it inhibits osteogenic differentiation of aortic valve interstitial cells. We demonstrated that SIRT6 was markedly downregulated in calcific human aortic valves. Mechanistically, SIRT6 suppressed osteogenic differentiation in human aortic valve interstitial cells (HAVICs), as confirmed by loss- and gain-of-function experiments. SIRT6 directly interacted with Runx2, decreased Runx2 acetylation levels, and facilitated Runx2 nuclear export to inhibit the osteoblastic phenotype transition of HAVICs. In addition, the AKT signaling pathway acted upstream of SIRT6. Together, these findings elucidate that SIRT6-mediated Runx2 downregulation inhibits aortic valve calcification and provide novel insights into therapeutic strategies for CAVD.

Outcomes of patients with early calcific aortic valve disease detected by clinically indicated echocardiography.

AIMS: Previous studies have demonstrated relatively slow rates of progression of early calcific aortic valve disease (CAVD), which encompasses aortic sclerosis (ASc) and mild aortic stenosis (AS). The potential evolution to clinically significant AS is unclear, and we therefore examined the long-term outcomes of patients with ASc and mild AS detected at the time of clinically indicated echocardiography. METHODS AND RESULTS: Data from initial clinically indicated echocardiograms performed between 2010 and 2018 in patients aged ≥18 years were extracted and linked to nationally collected outcome data. Those with impaired right or left systolic ventricular function or other significant left-sided valve disease were excluded. A time to first event analysis was performed with a composite primary outcome of cardiovascular death and aortic valve intervention (AVI). Of the 13 313 patients, 8973 had no CAVD, 3436 had ASc, and 455 had mild AS. The remainder had moderate or worse stenosis. Over a median follow-up period of 4.2 (interquartile range 1.8-6.7) years (and after adjustment for age and sex), those with ASc were at greater risk of the primary outcome [hazard ratio (HR) 2.9, 95% confidence interval (CI) 2.1-4.0] and need for AVI (HR 26.8, 95% CI 9.1-79.1) compared with those with no CAVD. Clinical event rates accelerated after ∼5 years in those with mild AS. CONCLUSION: Patients with ASc are >25 times more likely to require AVI than those with no CAVD, and follow-up echocardiography should be considered within 3-4 years in those with mild AS.

Plasma Cell-Free DNA Is a Potential Biomarker for Diagnosis of Calcific Aortic Valve Disease.

INTRODUCTION: Calcific aortic valve disease (CAVD) is the third most common cardiovascular disease in aging populations. Despite a growing number of biomarkers having been shown to be associated with CAVD, a marker suitable for routine testing in clinical practice is still needed. Plasma cell-free DNA (cfDNA) has been suggested as a biomarker for diagnosis and prognosis in multiple diseases. In this study, we aimed to test whether cfDNA could be used as a biomarker for the diagnosis of CAVD. METHODS: Serum samples were collected from 137 diagnosed CAVD patients and 180 normal controls. The amount of cfDNA was quantified by amplifying a short fragment (ALU 115) and a long fragment (ALU 247) using quantitative real-time PCR. The cfDNA integrity (cfDI) was calculated as the ratio of ALU247 to ALU115. The association between CAVD and cfDI was evaluated using regression analysis. RESULTS: CAVD patients had increased ALU 115 fragments (median, 185.14 (416.42) versus 302.83 (665.41), p < 0.05) but a decreased value of cfDI (mean, 0.50 ± 0.25 vs. 0.41 ± 0.26, p < 0.01) in their serum when compared to controls. This difference was more dramatic in non-rheumatic CAVD patients (p < 0.001) versus rheumatic CAVD patients (no significant difference). Similarly, CAVD patients with bicuspid aortic valve (BAV) (p < 0.01) showed a greater difference than non-BAV CAVD patients (p < 0.05). Linear regression and logistic regression showed that cfDI was independently and significantly associated with the presence of CAVD (95% CI, 0.096 to 0.773, p < 0.05). The ROC assay revealed that cfDI combined with clinical characteristics had a better diagnostic value than cfDI alone (AUC = 0.6191, p < 0.001). CONCLUSION: cfDI may be a potential biomarker for diagnosis of CAVD.

Long non-coding RNA FGD5 antisense RNA 1 targets Baculovirus inhibitor 5 via microRNA-497-5p to alleviate calcific aortic valve disease.

Calcific aortic valve disease (CAVD) is featured by thickening and calcification of the aortic valve. Osteoblast differentiation is a crucial step in valve calcification. Long non-coding RNAs (LncRNAs) participate in the osteogenic differentiation of mesenchymal cells. However, the character of lncRNA FGD5 antisense RNA 1 (FGD5-AS1) in CAVD is uncertain. After collection of human aortic valve tissue samples, detection of FGD5-AS1, microRNA (miR)-497-5p and Baculovirus inhibitor 5 (BIRC5) was conducted. Valve mesenchymal cells were isolated from CAVD patients and induced to differentiate to osteoblasts, and transfected with FGD5-AS1, miR-497-5p and BIRC5 plasmids. Detection of the alkaline phosphatase activity was after osteogenic induction of human aortic valve interstitial cells (hAVICs); Detection of the degree of calcium nodules and osteoblast differentiation markers (RUNX2 and OPN) was conducted. After establishment of a mouse model of CAVD, detection of the thickness of aortic valve leaflets, and the degree of calcification of the valve leaflets, and evaluation of echocardiographic parameters were implemented. Experimental data manifested in CAVD patients, lncRNAFGD5-AS1 and BIRC5 were reduced, but miR-497-5p was elevated; Enhancing lncRNA FGD5-AS1 or repressing miR-497-5p mitigated CAVD by restraining osteogenic differentiation; LncRNA FGD5-AS1 sponged miR-497-5p to target BIRC5; Repressive BIRC5 turned around the therapeutic action of elevated FGD5-AS1 or depressed miR-497-5p on hAVICs; Enhancive FGD5-AS1 in vivo was available to reduce ApoE-/- mouse CAVD induced via high cholesterol diet. All in all, lncRNAFGD5-AS1 targets BIRC5 via miR-497-5p to alleviate CAVD.