Calcific Aortic Valve Disease Research Articles

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.

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

BackgroundTransient 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). MethodsThe 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. ResultsThe 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. ConclusionThe 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.

#1409 Enhancer of zeste homolog 2 (EZH2) as a new therapeutic target to prevent indoxyl-sulfate-induced aortic valve calcification

Abstract Background and Aims Calcific aortic valve disease (CAVD) is highly prevalent in chronic kidney disease (CKD) patients and is associated with a poor prognosis. To date, there is no pharmacological treatment to slow down this process. Our group recently reported that indoxyl-sulfate (IS) -induced IL-6 secretion in human interstitial valvular cells (hVICs) promotes their osteogenic transition and calcification in an autocrine manner. Preliminary data suggest that IS also influences macrophage secretion of IL-6. Whether this phenomenon affects the hVICs mineralization remains to be elucidated. In this context, the epigenetic enzyme Enhancer of Zeste Homolog 2 (EZH2), a key modulator of macrophages’ inflammation, may represent an interesting therapeutic target. Thus, this work aimed to verify whether IS-induced macrophages’ secretion of IL-6 modulates hVICs mineralization, and to determine whether EZH2 is involved in this inflammatory process. Method THP1-derived macrophages were exposed to increasing concentrations of IS (IS normal [Isn]: 0.5 μg/mL, IS uremic [Isu]: 37 μg/mL, IS intermediate [int]: 100 μg/mL or IS maximum [Ismax]: 233 μg/mL) in the presence or absence of an EZH2 inhibitor called GSK-343 (5µM). EZH2 expression and activity (evidenced by the trimethylation of the lysine 27 of the histones H3 (H3K27me3)) were assessed by western blot. Macrophages’ inflammatory potential was checked by RT-qPCR, western blot and Elisa. The impact of macrophages exposed or not to IS and/or GSK-343 on hVICs osteogenic transition was evaluated using conditioned media. HVICs osteogenic transition was assessed following runx2 expression by qRT-PCR and western blot. Mineralization was quantified by the o-cresolphthalein method. Results IS induced macrophages secretion of IL-1β, IL-6, CCL2 and TNF-α. Conditioned media from IS-polarized macrophages promoted a 6-fold increase in hVICs calcification and favored their osteogenic transition as evidenced by increased runx2 expression. IL-6 was the only cytokine able to promote runx2 expression in hVICs, confirming the importance of this specific cytokine in the process. Exposure to IS upregulated macrophages’ expression EZH2, an effect blocked by GSK-343. If GSK-343 did not affect IS-induced macrophages’ secretion of IL-1β, CCL2 and TNF-α, it efficiently blocked the secretion of IL-6 and subsequent induction of runx2 in hVICs. In this model, neither IS, nor GSK-343 modulated macrophages’ H3K27me3, suggesting that EZH2 may act as a transcriptional factor for IL-6 through its non-canonical pathway. In this latter, EZH2 is thought to promote inflammation by binding to p65, rather than through its trimethylation activity. In line with this hypothesis, exposure to IS promoted EZH2 and p65 nuclear translocation in the meantime as well as their co-immunoprecipitation, two phenomena blocked by GSK343. Conclusion This work demonstrates that IS-induced macrophages’ secretion of IL-6 promotes hVICs osteogenic transition and mineralization. Our data suggest that IL-6 secretion in response to IS depends on the EZH2-p65 pathway. The fact that GSK-343 can block this mechanism sheds light on EZH2 as a new therapeutic target to prevent CAVD in CKD patients.

Deep-learning survival analysis for patients with calcific aortic valve disease undergoing valve replacement

Calcification of the aortic valve (CAVDS) is a major cause of aortic stenosis (AS) leading to loss of valve function which requires the substitution by surgical aortic valve replacement (SAVR) or transcatheter aortic valve intervention (TAVI). These procedures are associated with high post-intervention mortality, then the corresponding risk assessment is relevant from a clinical standpoint. This study compares the traditional Cox Proportional Hazard (CPH) against Machine Learning (ML) based methods, such as Deep Learning Survival (DeepSurv) and Random Survival Forest (RSF), to identify variables able to estimate the risk of death one year after the intervention, in patients undergoing either to SAVR or TAVI. We found that with all three approaches the combination of six variables, named albumin, age, BMI, glucose, hypertension, and clonal hemopoiesis of indeterminate potential (CHIP), allows for predicting mortality with a c-index of approximately 80%\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$80\\%$$\\end{document}. Importantly, we found that the ML models have a better prediction capability, making them as effective for statistical analysis in medicine as most state-of-the-art approaches, with the additional advantage that they may expose non-linear relationships. This study aims to improve the early identification of patients at higher risk of death, who could then benefit from a more appropriate therapeutic intervention.

Hyperglycemia-simulating environment attenuated experimentally induced calcification in cultured human aortic valve interstitial cells

Objectives: The role of diabetes mellitus as a risk factor for the development of calcific aortic valve disease has not been fully clarified. Aortic valve interstitial cells (VICs) have been suggested to be crucial for calcification of the valve. Induced calcification in cultured VICs is a good in vitro model for aortic valve calcification. The purpose of this study was to investigate whether increased glucose levels increase experimentally induced calcification in cultured human VICs. Design: VICs were isolated from explanted calcified aortic valves after valve replacement. Osteogenic medium induced calcification of cultured VICs at different glucose levels (5, 15, and 25 mM). Calcium deposits were visualized using Alizarin Red staining and measured spectrophotometrically. Results: The higher the glucose concentration, the lower the level of calcification. High glucose (25 mM) reduced calcification by 52% compared with calcification at a physiological (5 mM) glucose concentration (correlation and regression analysis: r = −0.55, p = .025 with increased concentration of glucose). Conclusions: In vitro hyperglycemia-like conditions attenuated calcification in VICs. High glucose levels may trigger a series of events that secondarily stimulate calcification of VICs in vivo.

The Relationship of Mitral Annular Calcification and Aortic Valve Sclerosis to Endothelial Dysfunction.

Calcific aortic valve disease (CAVD) and mitral annular calcification (MAC) are associated with various cardiovascular diseases and may influence systemic vascular pathologies. However, their relationship with endothelial dysfunction and carotid intima-media thickness (CIMT) remains poorly elucidated. This research aims to explore the associations between MAC, aortic valve sclerosis (AVS), and markers of vascular dysfunction, specifically CIMT and endothelial function. This prospective observational study included 200 patients undergoing routine echocardiographic evaluation at the National Heart Institute between May 2022 and April 2023. Patients were stratified into four groups namely isolated MAC (38 patients), isolated AVS (72 patients), combined MAC and AVS (50 patients), and a control group without MAC or AVS (40 patients). All participants underwent comprehensive cardiovascular evaluation, including transthoracic echocardiography (TTE) and carotid duplex ultrasonography. Endothelial function was determined by measuring reactive hyperemia-induced alterations in brachial artery diameter. The mean age of participants was 60.6±8.4 years, with a predominance of male subjects (64%). No significant differences were noted in baseline demographic and clinical characteristics across the groups. Patients with isolated AVS, isolated MAC, and both conditions demonstrated increased CIMT compared to controls, with significant differences noted in the combined MAC and AVS group compared to controls (p-value=0.031). Endothelial dysfunction was observed in 14.8% of the AVS group and 21.1% in the combined group, but no significant differences existed when compared to controls. The study also revealed that patients with AVS are more likely to exhibit increased CIMT (p-value=0.008). Both MAC and AVS are connected to increased CIMT, suggesting a link with systemic atherosclerotic processes. Although the existence of endothelial dysfunction was not significantly higher in patients with valvular calcifications, the findings support the need for further research into the cardiovascular implications of CAVD and MAC.

Abstract 3062: Activation Of Adam17-dlk1 Axis Induces Aortic Valve Calcification.

Aortic valve stenosis (AS) is characterized by valve architecture disruption through mineralization and valve cusps fibrosis. Currently, there are no proven pharmacotherapies to prevent or decelerate AS progression. Understanding the molecular mechanisms leading to aortic valve mineralization is of inestimable importance. A balance between calcium deposition and regression may be involved in the pathogenesis of calcified aortic valve disease. Delta-like 1 homolog (Dlk1) is an imprinted gene that can be proteolytically cleaved within the juxta-membrane region by TACE, resulting in the release of an extracellular part of Dlk1 as a soluble fragment known as fetal antigen 1 (FA1). DLK1 is associated with increased adiposity and fibrosis. Objective: extracellular matrix (ECM) remodeling disorder in response to TACE-Dlk1 defect might affect valve structure through ECM-lipid interaction in patients with abdominal obesity. We investigated the potential role of TACE/DLK1 in activating osteoblastic differentiation in mouse valve interstitial cells (VICs) in vitro. Method: We first isolated VICs from WT and DLK1-/- mice and treated them with a calcifying medium for seven days. Mass spectrometry examined extracellular protein expression (ECM) and increased lipid retention. Results: The loss of function of Dlk1 was associated with disrupted ECM expression and the increase of osteogenic genes (RUNX2, SPP1, BMP2, MSX2) expression. Conclusion: Dlk1 affects ECM expression and lipid retention, enhancing aortic valve degeneration.

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.

Association between lipoprotein(a), LPA genetic risk score, aortic valve disease, and subsequent major adverse cardiovascular events.

Cohort studies have demonstrated associations between calcific aortic valve disease (CAVD) and Lp(a). As Lp(a) is almost entirely genetically determined, in this study, we aim to determine whether Lp(a), when predicted from genetic data, is associated with CAVD and major adverse cardiovascular events (MACEs). Patients undergoing coronary angiography between January 2012 and May 2013 were invited to participate in the study. Of 752 analysable participants, 446 had their Lp(a) measured and 703 had a calculable LPA genetic risk score (GRS). The primary outcomes were the presence of CAVD at baseline and MACE over a 7-year follow-up. The GRS explained 45% of variation in Lp(a). After adjustment for cardiac risk factors and coronary artery disease (CAD), the odds of CAVD increased with increasing Lp(a) [odds ratio (OR) 1.039 per 10-unit increase, 95% confidence interval (CI) 1.022-1.057, P < 0.001] and GRS (OR 1.054 per 10-unit increase, 95% CI 1.024-1.086; P < 0.001). Lipoprotein(a) and the GRS as continuous variables were not associated with subsequent MACEs. A dichotomized GRS (>54) was associated with MACE, but this relationship became non-significant when CAD classification was added into the model (OR 1.333, 95% CI 0.927-1.912; P = 0.12). An LPA GRS can explain 45% of variation in Lp(a) levels, and both Lp(a) and the GRS are associated with CAVD. An elevated GRS is associated with future cardiac events in a secondary risk setting, but, if the CAD status is known, it does not provide additional prognostic information.

Integrated bioinformatics analysis identified leucine rich repeat containing 15 and secreted phosphoprotein 1 as hub genes for calcific aortic valve disease and osteoarthritis.

Calcific aortic valve disease (CAVD) and osteoarthritis (OA) are common diseases in the ageing population and share similar pathogenesis, especially in inflammation. This study aims to discover potential diagnostic and therapeutic targets in patients with CAVD and OA. Three CAVD datasets and one OA dataset were obtained from the Gene Expression Omnibus database. We used bioinformatics methods to search for key genes and immune infiltration, and established a ceRNA network. Immunohistochemical staining was performed to verify the expression of candidate genes in human and mice aortic valve tissues. Two key genes obtained, leucine rich repeat containing 15 (LRRC15) and secreted phosphoprotein 1 (SPP1), were further screened using machine learning and verified in human and mice aortic valve tissues. Compared to normal tissues, the infiltration of immune cells in CAVD tissues was significantly higher, and the expressions of LRRC15 and SPP1 were positively correlated with immune cells infiltration. Moreover, the ceRNA network showed extensive regulatory interactions based on LRRC15 and SPP1. The authors' findings identified LRRC15 and SPP1 as hub genes in immunological mechanisms during CAVD and OA initiation and progression, as well as potential targets for drug development.

343 Development of osteoclast derived exosomes for vascular calcification therapy

OBJECTIVES/GOALS: The global incidence of calcific aortic valve disease (CAVD) increased 3.5-fold since 1990. No preventative or therapeutic pharmaceutical therapies exist for CAVD. We will establish the therapeutic potential of osteoclast-derived exosomes though characterization of contents and mechanisms of action to protect against mineralization. METHODS/STUDY POPULATION: Exosomes were purified from conditioned media collected from murine myeloid precursor cells, RAW264.7 (control), and osteoclasts induced to differentiate from RAW264.7 cells (OD). Protein content of exosomes was determined using proteomic analyses. Nucleic acid contents will be identified by sequencing mRNA, miRNA, and DNA. The calcification prevention and reabsorption abilities of control and OD exosomes will be tested using human valvular interstitial cells (VIC) and smooth muscle cell calcification assays and acellular osteologic disc assays, respectively. Comparison between cellular and acellular systems will help identify mechanisms of action, and demonstrate potential therapeutic viability of OD exosomes in preventative vs resorptive treatments. RESULTS/ANTICIPATED RESULTS: OD exosomes, but not control exosomes, prevented calcification in VIC in vitro. OD exosomes contained osteoclast-specific proteins including TRAP, MMP6, cathepsin K, and bone reabsorption factors including V type proton pumps, ATPases, and integrins. These genes are also involved in resorptive activities, and were highly upregulated in OD compared to control exosomes. We anticipate miRNA signatures associated with mineral resorption will also be present. Increased knowledge of exosome cargo will illuminate their mechanism of action and allow future work to engineer increased efficacy. We also anticipate a therapeutic response when OD exosomes are applied after calcification has begun, showing exosomes promote calcium reabsorption. DISCUSSION/SIGNIFICANCE: Establishing therapeutic potential and examining mechanisms of action will pave the way for OD exosomes as a CAVD treatment. Analysis of exosome contents will determine active molecules to be enhanced in future studies. This work will lay a foundation for moving into aortic valve organoid models, which are accepted by the FDA for preclinical trials.

Investigation of autophagy‑related 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.

Indoxyl Sulfate-Induced Valve Endothelial Cell Endothelial-to-Mesenchymal Transition and Calcification in an Integrin-Linked Kinase-Dependent Manner.

Calcific Aortic Valve Disease (CAVD) is a significant concern for cardiovascular health and is closely associated with chronic kidney disease (CKD). Aortic valve endothelial cells (VECs) play a significant role in the onset and progression of CAVD. Previous research has suggested that uremic toxins, particularly indoxyl sulfate (IS), induce vascular calcification and endothelial dysfunction, but the effect of IS on valve endothelial cells (VECs) and its contribution to CAVD is unclear. Our results show that IS reduced human VEC viability and increased pro-calcific markers RUNX2 and alkaline phosphatase (ALP) expression. Additionally, IS-exposed VECs cultured in pro-osteogenic media showed increased calcification. Mechanistically, IS induced endothelial-to-mesenchymal transition (EndMT), evidenced by the loss of endothelial markers and increased expression of mesenchymal markers. IS triggered VEC inflammation, as revealed by NF-kB activation, and decreased integrin-linked kinase (ILK) expression. ILK overexpression reversed the loss of endothelial phenotype and RUNX2, emphasizing its relevance in the pathogenesis of CAVD in CKD. Conversely, a lower dose of IS intensified some of the effects in EndMT caused by silencing ILK. These findings imply that IS affects valve endothelium directly, contributing to CAVD by inducing EndMT and calcification, with ILK acting as a crucial modulator.

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.

Intracellular proteomics and extracellular vesiculomics as a metric of disease recapitulation in 3D-bioprinted aortic valve arrays.

In calcific aortic valve disease (CAVD), mechanosensitive valvular cells respond to fibrosis- and calcification-induced tissue stiffening, further driving pathophysiology. No pharmacotherapeutics are available to treat CAVD because of the paucity of (i) appropriate experimental models that recapitulate this complex environment and (ii) benchmarking novel engineered aortic valve (AV)-model performance. We established a biomaterial-based CAVD model mimicking the biomechanics of the human AV disease-prone fibrosa layer, three-dimensional (3D)-bioprinted into 96-well arrays. Liquid chromatography-tandem mass spectrometry analyses probed the cellular proteome and vesiculome to compare the 3D-bioprinted model versus traditional 2D monoculture, against human CAVD tissue. The 3D-bioprinted model highly recapitulated the CAVD cellular proteome (94% versus 70% of 2D proteins). Integration of cellular and vesicular datasets identified known and unknown proteins ubiquitous to AV calcification. This study explores how 2D versus 3D-bioengineered systems recapitulate unique aspects of human disease, positions multiomics as a technique for the evaluation of high throughput-based bioengineered model systems, and potentiates future drug discovery.

Mechanoregulation and function of calponin and transgelin.

It is well known that chemical energy can be converted to mechanical force in biological systems by motor proteins such as myosin ATPase. It is also broadly observed that constant/static mechanical signals potently induce cellular responses. However, the mechanisms that cells sense and convert the mechanical force into biochemical signals are not well understood. Calponin and transgelin are a family of homologous proteins that participate in the regulation of actin-activated myosin motor activity. An isoform of calponin, calponin 2, has been shown to regulate cytoskeleton-based cell motility functions under mechanical signaling. The expression of the calponin 2 gene and the turnover of calponin 2 protein are both under mechanoregulation. The regulation and function of calponin 2 has physiological and pathological significance, as shown in platelet adhesion, inflammatory arthritis, arterial atherosclerosis, calcific aortic valve disease, post-surgical fibrotic peritoneal adhesion, chronic proteinuria, ovarian insufficiency, and tumor metastasis. The levels of calponin 2 vary in different cell types, reflecting adaptations to specific tissue environments and functional states. The present review focuses on the mechanoregulation of calponin and transgelin family proteins to explore how cells sense steady tension and convert the force signal to biochemical activities. Our objective is to present a current knowledge basis for further investigations to establish the function and mechanisms of calponin and transgelin in cellular mechanoregulation.

Andrographolide regulates H3 histone lactylation by interfering with p300 to alleviate aortic valve calcification.

Our previous studies have found that andrographolide (AGP) alleviates calcific aortic valve disease (CAVD), but the underlying mechanism is unclear. This study explores the molecular target and signal mechanisms of AGP in inhibiting CAVD. The anti-calcification effects of the aortic valve with AGP treatment were evaluated by alizarin red staining in vitro and ultrasound and histopathological assessment of a high-fat (HF)-fed ApoE-/- mouse valve calcification model. A correlation between the H3 histone lactylation (H3Kla) and calcification was detected. Molecular docking and surface plasmon resonance (SPR) experiments were further used to confirm p300 as a target for AGP. Overexpression (oe) and silencing (si) of p300 were used to verify the inhibitory effect of AGP targeting p300 on the H3Kla in vitro and ex vivo. AGP significantly inhibited calcium deposition in valve interstitial cells (VICs) and ameliorated aortic valve calcification. The multi-omics analysis revealed the glycolysis pathway involved in CAVD, indicating that AGP interfered with lactate production by regulating lactate dehydrogenase A (LDHA). In addition, lactylation, a new post-translational modification, was shown to have a role in promoting aortic valve calcification. Furthermore, H3Kla and H3K9la site were shown to correlate with Runx2 expression inhibition by AGP treatment. Importantly, we found that p300 transferase was the molecular target of AGP in inhibiting H3Kla. Our findings, for the first time, demonstrated that AGP alleviates calcification by interfering with H3Kla via p300, which might be a powerful drug to prevent CAVD.

Biomechanical Remodeling of Aortic Valve Interstitial Cells During Calcified Lesion Formation In Vitro.

Healthy aortic heart valves are essential to the regulation of unidirectional blood flow. Calcific aortic valve disease (CAVD) is an actively progressive disease that involves the disorganization of valve cells and accumulation of calcium deposits on the aortic valve leaflets. CAVD involves disruption of cell environment homeostasis that prior cell culture models have found difficult to portray and model. As it is still poorly understood how tissue stiffening associates with lesion formation, here, we implement a novel 3D culture platform to characterize the relationship between mechanical stress and tissue remodeling and analyze how the application of pro-osteogenic stimulation dysregulates the native ability of valve cells to organize its matrix. Through a temporal study of macroscopic remodeling, we determine that aortic valve interstitial neo-tissues undergo varying stiffness and mechanical stress, demonstrate greater myofibroblastic gene expression, and show greater remodeling activity in the outer surface of the neo-tissue in a banding pattern when cultured in osteogenic growth medium. In human aortic valve interstitial cells cultured in osteogenic growth medium, we observed an increase in stress but significant decreases in myofibroblastic gene expression with the addition of growth factors. In summary, we are able to see the interplay of biochemical and biomechanical stimuli in valvular remodeling by using our platform to model dynamic stiffening of valve interstitial neo-tissues under different biochemical conditions.

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.