Hypericum monogynum extract inhibits human aortic valve interstitial cell calcification by interfering with the EGFR/PI3K/AKT signaling pathway.

The involvement of gut microbiota in calcific aortic valve disease (CAVD) pathogenesis remains underexplored. Here, we provide evidence for a strong association between the gut microbiota and CAVD development. ApoE−/− mice were stratified into easy‐ and difficult‐ to calcify groups using neural network and cluster analyses, and subsequent faecal transplantation and dirty cage sharing experiments demonstrated that the microbiota from difficult‐to‐calcify mice significantly ameliorated CAVD. 16S rRNA sequencing revealed that reduced abundance of Faecalibacterium prausnitzii (F. prausnitzii) was significantly associated with increased calcification severity. Association analysis identified F. prausnitzii‐derived butyric acid as a key anti‐calcific metabolite. These findings were validated in a clinical cohort (25 CAVD patients vs. 25 controls), where serum butyric acid levels inversely correlated with disease severity. Functional experiments showed that butyric acid effectively hindered osteogenic differentiation in human aortic valve interstitial cells (hVICs) and attenuated CAVD progression in mice. Isotope labeling and 13C flux analyses confirmed that butyric acid produced in the intestine can reach heart tissue, where it reshapes glycolysis by specifically modifying GAPDH. Mechanistically, butyric acid‐induced butyrylation (Kbu) at lysine 263 of GAPDH competitively inhibited lactylation (Kla) at the same site, thereby counteracting glycolysis‐driven calcification. These findings uncover a novel mechanism through which F. prausnitzii and its metabolite butyric acid contribute to the preservation of valve function in CAVD, highlighting the gut microbiota‐metabolite‐glycolysis axis as a promising therapeutic target.

Transcatheter aortic valve replacement valve-in-valve: Future implications for the surgeon

Calcific aortic valve disease (CAVD) is characterized by progressive stiffening of aortic valve (AV) tissues, inducing stenosis and insufficiency. Bicuspid aortic valve (BAV) is a common congenital defect in which the AV has two leaflets rather than three, with BAV patients developing CAVD decades years earlier than in the general population. Current treatment for CAVD remains surgical replacement with its continued durability problems, as there are no pharmaceutical therapies or other alternative treatments available. Before such therapeutic approaches can be developed, a deeper understanding of CAVD disease mechanisms is clearly required. It is known that AV interstitial cells (AVICs) maintain the AV extracellular matrix and are typically quiescent in the normal state, transitioning into an activated, myofibroblast-like state during periods of growth or disease. One proposed mechanism of CAVD is the subsequent transition of AVICs into an osteoblast-like phenotype. A sensitive indicator of AVIC phenotypic state is enhanced basal contractility (tonus), so that AVICs from diseased AV will exhibit a higher basal tonus level. The goals of the present study were thus to assess the hypothesis that different human CAVD states lead to different biophysical AVIC states. To accomplish this, we characterized AVIC basal tonus behaviors from diseased human AV tissues embedded in 3D hydrogels. Established methods were utilized to track AVIC-induced gel displacements and shape changes after the application of Cytochalasin D (an actin polymerization inhibitor) to depolymerize the AVIC stress fibers. Results indicated that human diseased AVICs from the non-calcified region of TAVs were significantly more activated than AVICs from the corresponding calcified region. In addition, AVICs from the raphe region of BAVs were more activated than from the non-raphe region. Interestingly, we observed significantly greater basal tonus levels in females compared to males. Furthermore, the overall AVIC shape changes after Cytochalasin suggested that AVICs from TAVs and BAVs develop different stress fiber architectures. These findings are the first evidence of sex-specific differences in basal tonus state in human AVICs in varying disease states. Future studies are underway to quantify stress fiber mechanical behaviors to further elucidate CAVD disease mechanisms.

Using bioinformatics analysis, network pharmacology, and cellular experiments, this study investigated the effects and potential mechanisms of Panax notoginseng saponins(PNS) on osteogenic induction of immortalized human aortic valve interstitial cells(hVICs). A calcific aortic valve disease(CAVD) cell model was established by treating hVICs with osteogenic induction medium(OM). Experimental groups included a control group, a model group, and PNS treatment groups(0.075, 0.05, and 0.025 mg·mL~(-1)). Cell viability was assessed by the CCK-8 assay. Calcification was evaluated by alkaline phosphatase(ALP) and alizarin red S staining. Western blot was performed to measure the expression of calcification-related proteins runt-related transcription factor 2(RUNX2), bone morphogenetic protein 2(BMP2), and ALP. Transcriptomic datasets related to CAVD were obtained from the Gene Expression Omnibus(GEO) database, and CAVD disease targets were retrieved from GeneCards. Active components of PNS and their potential targets were collected from HERB, Batman-TCM 2.0, and ETCM databases. The intersecting genes were subjected to protein-protein interaction(PPI) network construction, Gene Ontology(GO) enrichment, and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway analyses, identifying the spleen tyrosine kinase(SYK)/nuclear factor kappa-B(NF-κB) signaling pathway as a core pathway. Western blot was then used to assess the effects of PNS at the optimal concentration on SYK/NF-κB pathway-related proteins. NF-κB p65 nuclear translocation was examined by immunofluorescence co-localization, and ELISA was applied to measure tumor necrosis factor-α(TNF-α) and interleukin-10(IL-10) levels. RESULTS:: showed that PNS reduced alizarin red S and ALP staining in calcification-induced hVICs and downregulated calcification-related proteins BMP2, ALP, and RUNX2(P<0.05, P<0.01), with the most significant effect observed at 0.075 mg·mL~(-1). A total of 1 416 differentially expressed genes were identified from GEO, while 5 646 CAVD-related targets were obtained from GeneCards. From HERB, Batman-TCM 2.0, and ETCM, 605 PNS targets were collected, with 130 overlapping genes identified. These were mainly involved in inflammatory responses, lipopolysaccharide-mediated signaling, transcriptional regulation, protein phosphorylation, and NF-κB pathway activation, occurring primarily in membrane rafts and the golgi apparatus, with molecular functions such as kinase activity regulation and protein binding, and enriched in lipid metabolism, atherosclerosis, and NF-κB signaling pathways. Experimental validation showed that PNS significantly decreased NF-κB p65 expression(P<0.05), significantly increased IκBα expression(P<0.01), and markedly suppressed expression of p-SYK(P<0.01), p-IκBα(P<0.01), p-NF-κB p65(P<0.01), and p-NF-κB p50(P<0.01). PNS also significantly reduced NF-κB p65 nuclear/cytoplasmic fluorescence intensity(P<0.01), decreased TNF-α secretion in the supernatant(P<0.01), and promoted IL-10 secretion(P<0.01). In conclusion, PNS ameliorates hVICs calcification by downregulating IκBα, NF-κB p65, and NF-κB p50 expression, inhibiting phosphorylation of SYK, IκBα, NF-κB p65, and NF-κB p50, reducing NF-κB p65 nuclear translocation, and suppressing TNF-α expression.

Calcification and Thrombosis as Mediators of Bioprosthetic Valve Deterioration

Calcific aortic valve disease (CAVD) is a complex heart valve disease involving a wide range of pathological changes. Emerging evidence indicates that osteogenic differentiation of human aortic valve interstitial cells (hAVICs) plays a key role in valve calcification. In this study, we aimed to investigate the function of miR‐638 in hAVICs osteogenesis. Both miRNA microarray assay and qRT‐PCR results demonstrating miR‐638 was obviously up‐regulated in calcific aortic valves compared with non‐calcific valves. We also proved that miR‐638 was significantly up‐regulated during hAVICs osteogenic differentiation. Overexpression of miR‐638 suppressed osteogenic differentiation of hAVICs in vitro, whereas down‐regulation of miR‐638 enhance the process. Target prediction analysis and dual‐luciferase reporter assay confirmed that Sp7 transcription factor (Sp7) was a direct target of miR‐638. Furthermore, knockdown of Sp7 inhibited osteogenic differentiation of hAVICs, which is similar to the results observed in up‐regulation miR‐638. Our data indicated that miR‐638 plays an inhibitory role in hAVICs osteogenic differentiation, which may act by targeting Sp7. MiR‐638 may be a potential therapeutic target for CAVD.

Commentary: Coronary revascularization following aortic valve replacement: More than just a trivial event?

Calcific aortic valve disease (CAVD) is an active pathobiological process that takes place at the cellular and molecular levels. It involves fibrosis and calcification of aortic valve leaflets, which eventually contributes to heart failure. Galectin-3 (Gal-3), a β-galactoside-binding lectin, is involved in myocardial fibrosis and remodeling. Our study aimed to explore how Gal-3 promoted the osteogenic differentiation of human aortic valve interstitial cells (hVICs) along with elucidating the underlying molecular mechanisms. To determine the Gal-3 expression in this study, we included the blood samples and aortic valves (AVs) from patients with CAVD (n=20) and normal controls (n=20). The hVICs were stimulated by Osteogenic medium (OM) and were treated with or without recombinant human Gal-3. Calcified transformation of hVICs was assessed by Alizarin Red S staining and osteogenic gene/protein expression. RNA-sequencing was performed for all different treatments to investigate differentially expressed genes (DEGs) along with exploring the enriched pathways for potential molecular targets of Gal-3. The targets were further detected using Western blotting and immunofluorescence staining. Gal-3 levels were found to be significantly increased in CAVD patients. Treatment of valve interstitial cells (VICs) with Gal-3 led to a marked increase in Runx2 and ALP-mRNA/protein expression levels as well as calcification. Gene expression profiles of hVICs cultured with or without Gal-3 revealed 79 upregulated genes and 82 down-regulated genes, which were highly enriched in TNF and NF-κB signaling pathways. Furthermore, Gal-3 could activate the phosphorylation of IκBα and interfere with the translocation of p65 into the cell nucleus of hVICs. However, inhibition of this pathway can suppress the osteogenic differentiation by Gal-3. Gal-3 acts as a positive regulator of osteogenic differentiation by activating the NF-κB signaling pathway in hVICs. Our findings provide novel mechanistic insights into the critical role of Gal-3 in the CAVD progression.

Calcific aortic valve disease (CAVD) is a valvular disease frequently in the elderly individuals that can lead to the valve dysfunction. Osteoblastic differentiation of human aortic valve interstitial cells (HAVICs) induced by inflammation play a crucial role in CAVD pathophysiological processes. To date, no effective drugs for CAVD have been established, and new agents are urgently needed. Piericidin glycosides, obtained from a marine-derived Streptomyces strain, were revealed to have regulatory effects on mitochondria in previous studies. Here, we discovered that 13-hydroxypiericidin A 10-O-α-D-glucose (1→6)-β-D-glucoside (S18), a specific piericidin diglycoside, suppresses lipopolysaccharide- (LPS) induced inflammatory responses of HAVICs by alleviating mitochondrial stress in an interleukin (IL)-37-dependent manner. Knockdown of IL-37 by siRNA not only exaggerated LPS-induced HAVIC inflammation and mitochondrial stress but also abrogated the anti-inflammatory effect of S18 on HAVICs. Moreover, S18 alleviated aortic valve lesions in IL-37 transgenic mice of CAVD model. Microscale thermophoresis (MST) and docking analysis of five piericidin analogues suggested that diglycosides, but not monoglycosides, exert obvious IL-37-binding activity. These results indicate that S18 directly binds to IL-37 to alleviate inflammatory responses in HAVICs and aortic valve lesions in mice. Piericidin diglycoside S18 is a potential therapeutic agent to prevent the development of CAVD.

Introduction and objectives: Inflammation has been linked to metabolic reprogramming in several diseases, including cardiovascular pathogenesis. Calcific aortic valve disease (CAVD) is an increasingly prevalent valvulopathy, yet surgical replacement is the only available therapy. CAVD is characterized by a damaged endothelium, inflammation, exaggerated matrix remodeling, calcification, and metabolic changes. At the cellular level, recent evidence disclose the interplay between innate immunity/inflammatory pathways, i.e., Toll-like receptors (TLR)3/4 and interferon-γ receptor signaling, on the differentiation, calcification, and inflammation of valve interstitial cells (VIC) via stabilization of hypoxia-inducible factor (HIF)-1α. Given that this transcription factor is associated with metabolic reprogramming in several diseases, inflammation and metabolism may work in an interconnected manner in CAVD. Therefore, the main goal of this study was to investigate the metabolic reprogramming of VIC under inflammatory settings, and its contribution to the processes relevant to CAVD pathogenesis. Material and methods: Human VIC from patients with no valve disease were used as a model. To mimic an inflammatory environment, cells were treated with pro-inflammatory cytokines and pathogen patterns recognized by TLRs. Metabolic analysis was performed by real-time metabolic analysis using Seahorse extracellular flux assays, and [U-13C]-glucose tracing, by liquid chromatography/mass spectrometry. Metabolic gene profiles and metabolite production were evaluated by qPCR, Western blot, and commercial kits. Inflammation, calcification, and apoptosis were studied using Western blot, ELISA, qPCR, immunofluorescence, flow cytometry, gene silencing, and in vitro calcification assays. Validation of findings in human VIC was performed in quiescent VIC dedifferentiated from human VIC, in porcine 3D VIC-valve endothelial cell co-cultures, as well in valve leaflets and VIC explanted from patients with/without CAVD. Results: The main finding of the study is that inflammatory stimuli drive a metabolic reprogramming of VIC to a hyperglycolytic phenotype that mimics the metabolic phenotype in calcified valves. It is characterized by enhanced glycolysis and glycolytic ATP production, impaired pentose phosphate pathway (PPP), as well as damaged mitochondrial function with uncoupling of electron transport chain (ETC) and oxidative phosphorylation (OXPHOS). Furthermore, metabolic dysregulation is associated with reactive oxygen species (ROS) production, as well as increased reliance on glucose uptake for energy production and metabolite accumulation. Pharmacological approaches to metabolic routes demonstrate the role of glycolysis upregulation in processes relevant to CAVD, such as VIC differentiation, calcification, and inflammation, and have further highlight the contribution of PPP and oxidative stress in these processes. Our findings further reveal the involvement of the Janus kinase (JAK)-STAT/HIF-1α and nuclear factor (NF)-kB pathways in the metabolic reprogramming. Finally, the shift in VIC, also found in 3D VIC-VEC co-cultures exposed to inflammatory stimuli, replicates the hyperglycolytic profile of calcified cells and valve leaflets. Conclusion: Inflammation drives a metabolic shift in human VIC, mirroring the glycolytic phenotype in calcified valves, which is characterized by hyperglycolysis that is necessary to support inflammation, calcification, and osteogenic differentiation of VIC. Additional reprogramming of complementary catabolic pathways, such as PPP, tricarboxylic acid cycle, and oxidative phosphorylation, generates redox homeostasis alterations that further contribute to pathological processes in VIC. Thus, inflammation-triggered changes in metabolic phenotypes may play a relevant pathogenic role in the early stages of CAVD, and the identified metabolic routes may provide therapeutic clues for the disease.

Self-Expanding Versus Balloon-Expandable Valve: Are We at the Cusp of Delivering a Perfect Transcatheter Aortic Valve?

BackgroundCalcific aortic valve disease (CAVD) is the second leading cause of adult heart diseases. The purpose of this study is to investigate whether miR-101-3p plays a role in the human aortic valve interstitial cells (HAVICs) calcification and the underlying mechanisms.MethodsSmall RNA deep sequencing and qPCR analysis were used to determine changes in microRNA expression in calcified human aortic valves.ResultsThe data showed that miR-101-3p levels were increased in the calcified human aortic valves. Using cultured primary HAVICs, we demonstrated that the miR-101-3p mimic promoted calcification and upregulated the osteogenesis pathway, while anti-miR-101-3p inhibited osteogenic differentiation and prevented calcification in HAVICs treated with the osteogenic conditioned medium. Mechanistically, miR-101-3p directly targeted cadherin-11 (CDH11) and Sry-related high-mobility-group box 9 (SOX9), key factors in the regulation of chondrogenesis and osteogenesis. Both CDH11 and SOX9 expressions were downregulated in the calcified human HAVICs. Inhibition of miR-101-3p restored expression of CDH11, SOX9 and ASPN and prevented osteogenesis in HAVICs under the calcific condition.ConclusionmiR-101-3p plays an important role in HAVIC calcification through regulation of CDH11/SOX9 expression. The finding is important as it reveals that miR-1013p may be a potential therapeutic target for calcific aortic valve disease.

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 chronic, progressive inflammatory disease. Soluble extracellular matrix (ECM) proteins can function as damage-associated molecular patterns (DAMPs) and may play a role in the progression of CAVD. Matrilin-2 is an ECM protein and has been found to up-regulate the pro-osteogenic activity in human aortic valve interstitial cells (AVICs). Klotho is an anti-aging protein that is recently found to have an anti-inflammatory effect. The impact of matrilin-2 and Klotho on AVICs inflammatory response is unclear. This study is to test the hypothesis that matrilin-2 induces the inflammatory response in human AVICs and to explore the anti-inflammatory potential of Klotho for suppression AVIC inflammation. Methods and Results: Human AVICs isolated from normal valves were treated with recombinant matrilin-2 (2.0 μg/ml). Matrilin-2 caused NF-κB-dependent increase in the levels of ICAM-1, MCP-1 and IL-6. In addition, matrilin-2 induced rapid activation of PKR through Toll-like receptor (TLR) 2 and 4. Treatment with PKR inhibitors, 2-AP or C13H8N4OS, prior to matrilin-2 stimulation, abrogated NF-κB phosphorylation and intranuclear translocation. Inhibition of PKR abolished the production of inflammatory mediators induced by matrilin-2 in human AVICs. Further experiments using recombinant Klotho revealed that Klotho (0.5 μg/ml) suppressed the activation of PKR and NF-κB, and markedly reduced the production of inflammatory mediators in human AVICs exposed to matrilin-2. Conclusion: This study demonstrates that soluble matrilin-2 induces the inflammatory response in human AVICs through a TLR-PKR-NF-κB signaling cascade and that Klotho is capable of suppressing human AVICs inflammatory response to a soluble ECM protein. The novel findings of this study indicate that soluble ECM proteins may fuel the progression of CAVD by inducing aortic valve inflammation and that Klotho has the potential for suppression of such inflammation.

Aims: Calcific aortic valve disease (CAVD) is a chronic cardiovascular disease with high morbidity that lacks effective pharmacotherapeutics. As a natural flavonoid extracted from Ampelopsis grossedentata, dihydromyricetin (DHM) has been shown to be effective in protecting against atherosclerosis; yet, the therapeutic role of DHM in CAVD remains poorly understood. Herein, we aimed to clarify the therapeutic implications of DHM in CAVD and the underlying molecular mechanisms in human valvular interstitial cells (hVICs).Methods and Results: The protein levels of two known osteogenesis-specific genes (alkaline phosphatase, ALP; runt-related transcription factor 2, Runx2) and calcified nodule formation in hVICs were detected by Western blot and Alizarin Red staining, respectively. The results showed that DHM markedly ameliorated osteogenic induction medium (OM)–induced osteogenic differentiation of hVICs, as evidenced by downregulation of ALP and Runx2 expression and decreased calcium deposition. The SwissTargetPrediction database was used to identify the potential AVC-associated direct protein target of DHM. Protein–protein interaction (PPI) analysis revealed that c-KIT, a tyrosine-protein kinase, can act as a credible protein target of DHM, as evidenced by molecular docking. Mechanistically, DHM-mediated inhibition of c-KIT phosphorylation drove interleukin-6 (IL-6) downregulation in CAVD, thereby ameliorating OM-induced osteogenic differentiation of hVICs and aortic valve calcification progression.Conclusion: DHM ameliorates osteogenic differentiation of hVICs by blocking the phosphorylation of c-KIT, thus reducing IL-6 expression in CAVD. DHM could be a viable therapeutic supplement to impede CAVD.