Human Aortic Valve Research Articles

Inhibition of miR-101-3p prevents human aortic valve interstitial cell calcification through regulation of CDH11/SOX9 expression

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.

Complement Cross Talks With H-K-ATPase to Upregulate Runx2 in Human Aortic Valve Interstitial Cells

IntroductionCalcific aortic valve disease (CAVD) is a slowly progressive fibro-calcific valve leaflet disorder. The underlying pathophysiology is complex and not yet well understood. Complement is known to play a role in the pathogenesis of CAVD by upregulating Runx2 to induce profibrogenic change in human aortic valve interstitial cells (AVICs). Furthermore, H-K-ATPase has independently been shown to induce tissue calcification. Therefore, we hypothesized that complement cross talks with H-K-ATPase to upregulate Runx2 in human AVICs. Materials and methodsHuman AVICs were isolated from normal and calcified aortic valves. Cells were treated with a variation of complement, H-K-ATPase, or ERK1/2 inhibitors. H-K-ATPase and its association with complement in AVICs were investigated by reverse transcriptase–polymerase chain reaction, immunofluorescence, and Western blot. ResultsCalcified human AVICs expressed significantly higher H-K-ATPase level than normal human AVICs. Presence of complement C3 with H-K-ATPase is found in AVICs after complement treatment. Complement induced both H-K-ATPase and Runx2 expression in AVICs, which was associated with increased phosphorylation of ERK1/2 and its downstream molecule p-70 S6. Pharmacological inhibition of either H-K-ATPase or Erk1/2 abolished complement-induced Runx2 expression. ConclusionsThese findings indicate that complement cross talks with H-K-ATPase to upregulate Runx2 in human AVICs by activation of ERK1/2 signaling pathways. The study revealed the potential role of H-K-ATPase in the pathogenesis of CAVD and therapeutically targeting either complement system or H-K-ATPase may limit the development of CAVD.

Rho A/ROCK1 signaling-mediated metabolic reprogramming of valvular interstitial cells toward Warburg effect accelerates aortic valve calcification via AMPK/RUNX2 axis

The aberrant differentiation of valvular interstitial cells (VICs) to osteogenic lineages promotes calcified aortic valves disease (CAVD), partly activated by potentially destructive hemodynamic forces. These involve Rho A/ROCK1 signaling, a mechano-sensing pathway. However, how Rho A/ROCK1 signaling transduces mechanical signals into cellular responses and disrupts normal VIC homeostasis remain unclear. We examined Rho A/ROCK1 signaling in human aortic valves, and further detected how Rho A/ROCK1 signaling regulates mineralization in human VICs. Aortic valves (CAVD n = 22, normal control (NC) n = 12) from patients undergoing valve replacement were investigated. Immunostaining and western blotting analysis indicated that Rho A/ROCK1 signaling, as well as key transporters and enzymes involved in the Warburg effect, were markedly upregulated in human calcified aortic valves compared with those in the controls. In vitro, Rho A/ROCK1-induced calcification was confirmed as AMPK-dependent, via a mechanism involving metabolic reprogramming of human VICs to Warburg effect. Y-27632, a selective ROCK1 inhibitor, suppressed the Warburg effect, rescued AMPK activity and subsequently increased RUNX2 ubiquitin-proteasome degradation, leading to decreased RUNX2 protein accumulation in human VICs under pathological osteogenic stimulus. Rho A/ROCK1 signaling, which is elevated in human calcified aortic valves, plays a positive role in valvular calcification, partially through its ability to drive metabolic switching of VICs to the Warburg effect, leading to altered AMPK activity and RUNX2 protein accumulation. Thus, Rho A/ROCK1 signaling could be an important and unrecognized hub of destructive hemodynamics and cellular aerobic glycolysis that is essential to promote the CAVD process.

Hydrogen sulfide as an anti-calcification stratagem in human aortic valve: Altered biogenesis and mitochondrial metabolism of H2S lead to H2S deficiency in calcific aortic valve disease

Hydrogen sulfide (H2S) was previously revealed to inhibit osteoblastic differentiation of valvular interstitial cells (VICs), a pathological feature in calcific aortic valve disease (CAVD). This study aimed to explore the metabolic control of H2S levels in human aortic valves.Lower levels of bioavailable H2S and higher levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were detected in aortic valves of CAVD patients compared to healthy individuals, accompanied by higher expression of cystathionine γ-lyase (CSE) and same expression of cystathionine β-synthase (CBS). Increased biogenesis of H2S by CSE was found in the aortic valves of CAVD patients which is supported by increased production of lanthionine. In accordance, healthy human aortic VICs mimic human pathology under calcifying conditions, as elevated CSE expression is associated with low levels of H2S. The expression of mitochondrial enzymes involved in H2S catabolism including sulfide quinone oxidoreductase (SQR), the key enzyme in mitochondrial H2S oxidation, persulfide dioxygenase (ETHE1), sulfite oxidase (SO) and thiosulfate sulfurtransferase (TST) were up-regulated in calcific aortic valve tissues, and a similar expression pattern was observed in response to high phosphate levels in VICs. AP39, a mitochondria-targeting H2S donor, rescued VICs from an osteoblastic phenotype switch and reduced the expression of IL-1β and TNF-α in VICs. Both pro-inflammatory cytokines aggravated calcification and osteoblastic differentiation of VICs derived from the calcific aortic valves. In contrast, IL-1β and TNF-α provided an early and transient inhibition of VICs calcification and osteoblastic differentiation in healthy cells and that effect was lost as H2S levels decreased. The benefit was mediated via CSE induction and H2S generation.We conclude that decreased levels of bioavailable H2S in human calcific aortic valves result from an increased H2S metabolism that facilitates the development of CAVD. CSE/H2S represent a pathway that reverses the action of calcifying stimuli.

Herpud1 deficiency alleviates homocysteine-induced aortic valve calcification.

To evaluate the role and therapeutic value of homocysteine (hcy)-inducible endoplasmic reticulum stress (ERS) protein with ubiquitin like domain 1 (Herpud1) in hcy-induced calcific aortic valve disease (CAVD). The morbidity and mortality rates of calcific aortic valve disease (CAVD) remain high while treatment options are limited. In vivo, we use the low-density lipoprotein receptor (LDLR) and Herpud1 double knockout (LDLR-/-/Herpud1-/-) mice and used high methionine diet (HMD) to assess of aortic valve calcification lesions, ERS activation, autophagy, and osteogenic differentiation of aortic valve interstitial cells (AVICs). In vitro, the role of Herpud1 in the Hcy-related osteogenic differentiation of AVICs was investigated by manipulating of Herpud1 expression. Herpud1 was highly expressed in calcified human and mouse aortic valves as well as primary aortic valve interstitial cells (AVICs). Hcy increased Herpud1 expression through the ERS pathway and promoted CAVD progression. Herpud1 deficiency inhibited hcy-induced CAVD in vitro and in vivo. Herpud1 silencing activated cell autophagy, which subsequently inhibited hcy-induced osteogenic differentiation of AVICs. ERS inhibitor 4-phenyl butyric acid (4-PBA) significantly attenuated aortic valve calcification in HMD-fed low-density lipoprotein receptor-/- (LDLR-/-) mice by suppressing ERS and subsequent Herpud1 biosynthesis. These findings identify a previously unknown mechanism of Herpud1 upregulation in Hcy-related CAVD, suggesting that Herpud1 silencing or inhibition is a viable therapeutic strategy for arresting CAVD progression. • Herpud1 is upregulated in the leaflets of Hcy-treated mice and patients with CAVD. • In mice, global knockout of Herpud1 alleviates aortic valve calcification and Herpud1 silencing activates cell autophagy, inhibiting osteogenic differentiation of AVICs induced by Hcy. • 4-PBA suppressed Herpud1 expression to alleviate AVIC calcification in Hcy treated AVICs and to mitigate aortic valve calcification in mice.

Ultrastructural and Immunohistochemical Detection of Hydroxyapatite Nucleating Role by rRNA and Nuclear Chromatin Derivatives in Aortic Valve Calcification: In Vitro and In Vivo Pro-Calcific Animal Models and Actual Calcific Disease in Humans.

Calcification starts with hydroxyapatite (HA) crystallization on cell membranous components, as with aortic valve interstitial cells (AVICs), wherein a cell-membrane-derived substance containing acidic phospholipids (PPM/PPLs) acts as major crystal nucleator. Since nucleic acid removal is recommended to prevent calcification in valve biosubstitutes derived from decellularized valve scaffolds, the involvement of ribosomal RNA (rRNA) and nuclear chromatin (NC) was here explored in three distinct contexts: (i) bovine AVIC pro-calcific cultures; (ii) porcine aortic valve leaflets that had undergone accelerated calcification after xenogeneic subdermal implantation; and (iii) human aortic valve leaflets affected by calcific stenosis. Ultrastructurally, shared AVIC degenerative patterns included (i) the melting of ribosomes with PPM/PPLs, and the same for apparently well-featured NC; (ii) selective precipitation of silver particles on all three components after adapted von Kossa reactions; and (iii) labelling by anti-rRNA immunogold particles. Shared features were also provided by parallel light microscopy. In conclusion, the present results indicate that rRNA and NC contribute to AVIC mineralization in vitro and in vivo, with their anionic charges enhancing the HA nucleation capacity exerted by PPM/PPL substrates, supporting the concept that nucleic acid removal is needed for valve pre-implantation treatments, besides better elucidating the modality of pro-calcific cell death.

Recombinant IL-37 Exerts an Anti-inflammatory Effect on Human Aortic Valve Interstitial Cells through Extracellular and Intracellular Actions.

Calcific aortic valve disease (CAVD) is a chronic inflammatory disease with slow progression that involves soluble extracellular matrix (ECM) proteins. Previously, we found that recombinant interleukin (IL)-37 suppresses aortic valve interstitial cells (AVIC) inflammatory response through the interaction with IL-18 receptor α-chain (IL-18Rα) on the cell surface. Endogenous IL-37 can be retained in the cytoplasm or released into extracellular spaces. It remains unknown whether recombinant IL-37 exerts the anti-inflammatory effect through intracellular action. Here, we found that recombinant IL-37 suppressed AVIC inflammatory response to soluble ECM proteins. Interestingly, recombinant IL-37 was internalized by human AVICs in an IL-18Rα-independent fashion. Blocking endocytic pathways reduced the internalization and anti-inflammatory potency of recombinant IL-37. Overexpression of IL-37 in human AVICs suppressed soluble ECM proteins-induced NF-κB activation and the production of ICAM-1 and VCAM-1. However, IL-37D20A (mutant IL-37 lacking nucleus-targeting sequences) overexpression had no such effect, and the inflammatory response to soluble ECM proteins was essentially intact in AVICs from transgenic mice expressing IL-37D20A. Together, recombinant IL-37 can be internalized by human AVICs through endocytosis. Intracellular IL-37 exerts an anti-inflammatory effect through a nucleus-targeting mechanism. This study highlights the potent anti-inflammatory effect of recombinant IL-37 in both extracellular and intracellular compartments through distinct mechanisms.

Gli1 promotes the phenotypic transformation of valve interstitial cells through Hedgehog pathway activation exacerbating calcific aortic valve disease

Calcific aortic valve disease (CAVD) is the most prevalent human valve disease worldwide. Multiple factors induce "irreversible" pathological changes in the aortic valve leaflets, resulting in changes in cardiac hemodynamics, eventually leading to heart failure. However, no effective pharmaceutical interventions have been found and prosthetic valve replacement is the only curative approach. Glioma-associated oncogene 1 (Gli1) exerts a regulatory role on cardiovascular diseases, and it is already a therapeutic target to combat tumors. Our research aimed to explore the role and basic mechanism of Gli1 in CAVD, to pave the way for the discovery of effective drugs in the treatment of CAVD. Human aortic valve tissues were obtained to evaluate Gli1 expression and primary valve interstitial cells (VICs) were used to perform related experiments. The results showed that Gli1 promoted cell proliferation and significantly accelerated cell osteogenic transformation through the up-regulation of the osteogenic factors Runx2 and Alp, in turn through the AKT signaling pathway by targeting P130cas expression. Furthermore, Gli1 was activated by TGF-β and sonic hedgehog through the canonical and non-canonical Hedgehog signaling pathways in VICs. Our results indicated that Gli1 promoted cell proliferation and accelerated cell osteogenic transformation in VICs, providing a new strategy for the therapy of CAVD by targeting Gli1.

Potential biomarkers and immune cell infiltration involved in aortic valve calcification identified through integrated bioinformatics analysis.

Background: Calcific aortic valve disease (CAVD) is the most common valvular heart disease in the aging population, resulting in a significant health and economic burden worldwide, but its underlying diagnostic biomarkers and pathophysiological mechanisms are not fully understood. Methods: Three publicly available gene expression profiles (GSE12644, GSE51472, and GSE77287) from human Calcific aortic valve disease (CAVD) and normal aortic valve samples were downloaded from the Gene Expression Omnibus database for combined analysis. R software was used to identify differentially expressed genes (DEGs) and conduct functional investigations. Two machine learning algorithms, least absolute shrinkage and selection operator (LASSO) and support vector machine-recursive feature elimination (SVM-RFE), were applied to identify key feature genes as potential biomarkers for Calcific aortic valve disease (CAVD). Receiver operating characteristic (ROC) curves were used to evaluate the discriminatory ability of key genes. The CIBERSORT deconvolution algorithm was used to determine differential immune cell infiltration and the relationship between key genes and immune cell types. Finally, the Expression level and diagnostic ability of the identified biomarkers were further validated in an external dataset (GSE83453), a single-cell sequencing dataset (SRP222100), and immunohistochemical staining of human clinical tissue samples, respectively. Results: In total, 34 identified DEGs included 21 upregulated and 13 downregulated genes. DEGs were mainly involved in immune-related pathways such as leukocyte migration, granulocyte chemotaxis, cytokine activity, and IL-17 signaling. The machine learning algorithm identified SCG2 and CCL19 as key feature genes [area under the ROC curve (AUC) = 0.940 and 0.913, respectively; validation AUC = 0.917 and 0.903, respectively]. CIBERSORT analysis indicated that the proportion of immune cells in Calcific aortic valve disease (CAVD) was different from that in normal aortic valve tissues, specifically M2 and M0 macrophages. Key genes SCG2 and CCL19 were significantly positively correlated with M0 macrophages. Single-cell sequencing analysis and immunohistochemical staining of human aortic valve tissue samples showed that SCG2 and CCL19 were increased in Calcific aortic valve disease (CAVD) valves. Conclusion: SCG2 and CCL19 are potential novel biomarkers of Calcific aortic valve disease (CAVD) and may play important roles in the biological process of Calcific aortic valve disease (CAVD). Our findings advance understanding of the underlying mechanisms of Calcific aortic valve disease (CAVD) pathogenesis and provide valuable information for future research into novel diagnostic and immunotherapeutic targets for Calcific aortic valve disease (CAVD).

Abstract 13942: Circular RNA-HIPK3 Suppresses Aortic Valve Calcification by Dual Inhibition of the Wnt/β-catenin Pathway in Aortic Valve Interstitial Cells

Introduction: Calcific aortic valve disease (CAVD) has become an increasingly important worldwide medical problem without effective pharmacological intervention. Circular RNAs (circRNAs), a large class of non-coding RNAs, have been shown to play a critical role in the occurrence and progression of cardiovascular disease. However, the role of circRNAs in CAVD remains unclear. Methods and Results: High-throughput sequencing of circRNAs expression profiles in human calcified aortic valves and normal controls revealed that circRNA-HIPK3 (circHIPK3) was abundant in aortic valves but decreased in calcified aortic valves. Infecting the CAVD mouse model with adeno-associated virus subtype 9 expressing circHIPK3 (AAV9-circHIPK3) revealed that circHIPK3 overexpression reduced thickness and calcium deposition of the aortic valve leaflet in vivo . Gain- or loss-of-function experiments showed that circHIPK3 inhibited osteogenic responses in human aortic valve interstitial cells. MeRIP and RIP experiments showed that circHIPK3 enhanced the stability of suppressor of morphogenesis in genitalia 1 (SMG1) mRNA in an N6-methyladenosine (m 6 A)-dependent manner. Meanwhile, RNA pull-down and ChIP-Seq indicated that circHIPK3 increased the transcriptional activity of Chibby family member 1(CBY1) by promoting TATA-box binding protein-associated factor 15 (TAF15) intranuclear translocation. Furthermore, SMG1 and CBY1 negatively regulated the activity of Wnt/β-catenin pathway, and rescue experiments showed that circHIPK3 inhibited the Wnt/β-catenin pathway through increasing the expression of SMG1 and CBY1. Conclusions: Deficiency of circHIPK3 results in activation of the Wnt/β-catenin pathway and thereby induces aortic valve calcification. Exogenous circHIPK3 supplementation shows the ability to inhibit aortic valve thickening and calcification in vivo . Therefore, targeting circHIPK3 may be a potential strategy to prevent the development of CAVD.

Abstract 12839: Integrated Multi-Omics and Single Nucleus Transcriptomics Highlight Enhanced Adaptive Immune Responses in Human Bicuspid Aortic Valve Stenosis

Introduction: Bicuspid aortic valve (BAV) is the most common congenital cardiac defect, occurring in 1.5% of humans. Due to unknown pathogenic mechanisms, over 70% of those with BAV develop calcific aortic valve disease (CAVD). These patients require valve replacement 25x more frequently and a decade earlier than those with a tricuspid aortic valve (TAV). Objective: Identify cellular and molecular drivers of accelerated disease in BAV-CAVD. Methods: Small RNA-seq, RNA-seq, and proteomics were performed on 70 human aortic valves (11 non-diseased TAVs; fibrotic and calcific stages of 32 TAVs and 27 BAVs excised for CAVD). Single nuclei RNA-seq (snRNA-seq) was completed on 54,608 nuclei from 13 additional human valves (4 non-diseased TAVs; 5 TAVs and 4 BAVs with CAVD). Results: 4,938 miRs, transcripts, and proteins were differentially enriched between stages of BAV- and TAV-CAVD (q<0.05). Multi-omics data, CT-derived valve calcification, and 29 clinical parameters were integrated by latent factor analysis. Protein-protein interaction networks revealed significantly elevated adaptive immune responses in BAV-CAVD: T cell/B cell inflammatory activation was enhanced and SLIT-ROBO signaling disrupted in diseased BAVs. snRNA-seq identified 10 major cell types in human aortic valves: valvular interstitial cells (65% of cells; 3 states), endothelial cells (10%; 2), macrophages (13%), T cells (8%; 2), and B cells (3%; 2). CAVD drove differentiation of dual side-specific endothelial subpopulations, enrichment of CARMN/CACNA1C/ACTA2-high interstitial cells, and substantial immune cell accumulation. B cells were further enriched in BAV- vs. TAV-CAVD. Conclusions: Adaptive immunity underpins molecular differences in BAV- vs. TAV-CAVD and is a novel avenue for tailored pharmacotherapy. We potentiate targeting of key cellular subpopulations by defining the dissociation bias-free transcriptional and cellular diversity of human CAVD at single-cell resolution.

Abstract 13265: Soluble Klotho Captures FGF23 to Mitigate Aortic Valve Inflammocalcification Associated With Chronic Kidney Disease

Calcific aortic valve disease (CAVD) is prevalent in the elderly people, particularly those with chronic kidney disease (CKD). Klotho (KL), an anti-aging protein, is expressed mainly in the kidney and the cardiovascular system as soluble and membrane forms. CKD patients have reduced levels of KL and increased levels of fibroblast growth factor 23 (FGF23), a pro-inflammatory mediator that may interact with soluble KL. We have reported that KL reduces the osteogenic response to high phosphate levels, a condition seen in CKD, in human aortic valve interstitial cells (AVICs). This study tested the hypothesis that soluble KL captures FGF23 to prevent aortic valve inflammocalcification induced by CKD. Methods and Results: We developed a novel CKD-CAVD model in old C57 mice (20 months) on AIN-76A diet and treated with adenine and calcitriol. Histology and immunostaining revealed tubular damage and monocyte/macrophage accumulation in kidney tissue of CKD mice. Aortic valve lesions, thickening and calcification, were evident in CKD mice. Serum FGF23 levels were increased while serum KL levels decreased. In addition, KL expression in the aortic valve was reduced. Recombinant FGF23 (0.04 mg/ml) elevated the inflammosteogenic activity in human AVICs. Treatment with recombinant KL (0.5 μg/ml) markedly decreased FGF23-induced inflammosteogenic activation in AVICs. Systemic analysis revealed that FGF23 upregulates YAP/SMAD3 signaling, and inhibition of YAP and SMAD3 reduced the effect of FGF23 on the inflammosteogenic activity in AVICs. In addition, recombinant KL suppressed the activation of this signaling mechanism in AVICs, and treatment of CKD mice with recombinant KL attenuated aortic valve lesions. However, denaturation of KL protein resulted in a loss of its effect on FGF23-induced AVIC inflammosteogenic activation. Further, incubation of FGF23 with recombinant KL not only resulted in the formation of complexes of these two proteins, but also markedly decreased the pro-inflammosteogenic effect of FGF23 in AVICs. Conclusion: This study demonstrates that CKD induces KL insufficiency and FGF23 accumulation that promote aortic valve lesions. Recombinant KL is capable of binding to FGF23 to suppress its pro-inflammosteogenic effect in the aortic valve.

HIF1A inhibitor PX-478 reduces pathological stretch-induced calcification and collagen turnover in aortic valve.

HIF1A is significantly upregulated in calcified human aortic valves (AVs). Furthermore, HIF1A inhibitor PX-478 was shown to inhibit AV calcification under static and disturbed flow conditions. Since elevated stretch is one of the major mechanical stimuli for AV calcification, we investigated the effect of PX-478 on AV calcification and collagen turnover under a pathophysiological cyclic stretch (15%) condition. Porcine aortic valve (PAV) leaflets were cyclically (1 Hz) stretched at 15% for 24 days in osteogenic medium with or without PX-478. In addition, PAV leaflets were cyclically stretched at a physiological (10%) and 15% for 3 days in regular medium to assess its effect of on HIF1A mRNA expression. It was found that 100 μM (high concentration) PX-478 could significantly inhibit PAV calcification under 15% stretch, whereas 50 μM (moderate concentration) PX-478 showed a modest inhibitory effect on PAV calcification. Nonetheless, 50 μM PX-478 significantly reduced PAV collagen turnover under 15% stretch. Surprisingly, it was observed that cyclic stretch (15% vs. 10%) did not have any significant effect on HIF1A mRNA expression in PAV leaflets. These results suggest that HIF1A inhibitor PX-478 may impart its anti-calcific and anti-matrix remodeling effect in a stretch-independent manner.

An endogenous inhibitor of angiogenesis downregulated by hypoxia in human aortic valve stenosis promotes disease pathogenesis

Aortic valve stenosis is the most common valve disease in the western world. Central to the pathogenesis of this disease is the growth of new blood vessels (angiogenesis) within the aortic valve allowing infiltration of immune cells and development of intra-valve inflammation. Identifying the cellular mediators involved in this angiogenesis is important as this may reveal new therapeutic targets which could ultimately prevent the progression of aortic valve stenosis. Aortic valves from patients undergoing surgery for aortic valve replacement or dilation of the aortic arch were examined both ex vivo and in vitro. We now demonstrate that the anti-angiogenic protein, soluble fms-like tyrosine kinase 1 (sFlt1), a non-signalling soluble receptor for vascular endothelial growth factor, is constitutively expressed in non-diseased valves. sFlt-1 expression was, however, significantly reduced in aortic valve tissue from patients with aortic valve stenosis while protein markers of hypoxia were simultaneously increased. Exposure of primary-cultured valve interstitial cells to hypoxia resulted in a decrease in the expression of sFlt-1. We further reveal using a bioassay that siRNA knock-down of sFlt1 in valve interstitial cells directly results in a pro-angiogenic environment. Finally, incubation of aortic valves with sphingosine 1-phosphate, a bioactive lipid-mediator, increased sFlt-1 expression and inhibited angiogenesis within valve tissue. In conclusion, this study demonstrates that sFlt1 expression is directly correlated with angiogenesis in aortic valves and the observed decrease in sFlt-1 expression in aortic valve stenosis could increase valve inflammation, promoting disease progression. This could be a viable therapeutic target in treating this disease.

Identifying hub genes of calcific aortic valve disease and revealing the immune infiltration landscape based on multiple WGCNA and single-cell sequence analysis.

Calcific aortic valve disease (CAVD) is a progressive fibrocalcific disease that can be treated only through valve replacement. This study aimed to determine the role of hub genes and immune cell infiltration in CAVDprogression. In this study, bioinformatics analysis was used to identify hub genes involved in CAVD. The datasets were downloaded from the Gene Expression Omnibus (GEO) database. Gene expression differences were evaluated via pathway and Gene Ontology analyses. Weighted gene co-expression network analysis (WGCNA) and differentially expressed genes were used to screen hub genes. The CIBERSORT algorithm was used to compare immune infiltration into the calcified aortic valve based on the hub genes between high- and low-expression groups. We also performed single-cell RNA sequencing based on six different human aortic valve leaflets. The expression of hub genes was identified in human and mouse samples through quantitative real-time polymerase chain reaction (qPCR), immunohistochemistry, immunofluorescence, and ELISA, and clinical features of the patients wereinvestigated. In total, 454 differentially expressed genes were obtained from the GEO database. WGCNA was used to find 12 co-expression modules in the Array Express database, of which one hub module (brown module) was most correlated with CAVD. Two hub genes were identified after combining the differentially expressed genes S100A8 and S100A9. Regarding these genes, the immune infiltration profiles varied between high- and low-expression groups. Compared with that in the low hub gene expression group, the high hub gene expression group had a higher proportion of activated NK cells (p < 0.01) and M1 macrophages (p < 0.05). The expression of S100A8 and S100A9 was consistent with single-gene RNA sequencing results, confirming that the expression levels of these two hub genes are significantly upregulated in patients with CAVD (p < 0.01). Furthermore, these results were verified using mouse and human samples by performing immunofluorescence, immunohistochemistry, qPCR, and ELISA analyses. Finally, the localization of S100A8 and S100A9 in monocytes and macrophages was confirmed via immunofluorescence using human aortic valves. These results demonstrate that S100A8 and S100A9 are two hub genes involved in CAVD, which might play an important role in its development through immune-related signaling pathways.

Hesperetin, a Promising Dietary Supplement for Preventing the Development of Calcific Aortic Valve Disease.

No effective therapeutic agents for calcific aortic valve disease (CAVD) are available currently. Dietary supplementation has been proposed as a novel treatment modality for various diseases. As a flavanone, hesperetin is widely abundant in citrus fruits and has been proven to exert protective effects in multiple diseases. However, the role of hesperetin in CAVD remains unclear. Human aortic valve interstitial cells (VICs) were isolated from aortic valve leaflets. A mouse model of aortic valve stenosis was constructed by direct wire injury (DWI). Immunoblotting, immunofluorescence staining, and flow cytometry were used to investigate the roles of sirtuin 7 (Sirt7) and nuclear factor erythroid 2-related factor 2 (Nrf2) in hesperetin-mediated protective effects in VICs. Hesperetin supplementation protected the mice from wire-injury-induced aortic valve stenosis; in vitro, hesperetin inhibited the lipopolysaccharide (LPS)-induced activation of NF-κB inflammatory cytokine secretion and osteogenic factors expression, reduced ROS production and apoptosis, and abrogated LPS-mediated injury to the mitochondrial membrane potential and the decline in the antioxidant levels in VICs. These benefits of hesperetin may have been obtained by activating Nrf2-ARE signaling, which corrected the dysfunctional mitochondria. Furthermore, we found that hesperetin could directly bind to Sirt7 and that the silencing of Sirt7 decreased the effects of hesperetin in VICs and potently abolished the ability of hesperetin to increase Nrf2 transcriptional activation. Our work demonstrates that hesperetin plays protective roles in the aortic valve through the Sirt7-Nrf2-ARE axis; thus, hesperetin might be a potential dietary supplement that could prevent the development of CAVD.

Unravelling the role of SOCS3 in aortic valve stenosis

Abstract Background Calcific aortic valve stenosis (CAVD) is the most prevalent heart valve disease worldwide and, in advanced stages, leads to clinical deterioration and poor prognosis. Until now, no medical treatment is available. Thought to be a merely degenerative disease, we now know that disease initiation and progression are actively regulated by immune cell infiltration, chronic inflammation, osteogenic differentiation of valvular interstitial cells (VIC) and endothelial-to-mesenchymal transition (EndMT) of valvular endothelial cells (VEC). Suppressor of Cytokine Signaling 3 (SOCS3) is known to be a key regulator of inflammation. It promotes the polarisation of macrophages to an inflammatory phenotype and thus, contributes to disease progression in atherosclerosis. In CAVD, not only inflammation but also heterotopic bone formation leads to the thickening and obstruction of the aortic valve cusps and as SOCS3 is one of the main regulators of physiological bone formation, it could play a critical role in the progression of CAVD. Purpose We hypothesize that SOCS3 plays a crucial role during calcification of the aortic valve through regulation of EndMT, calcification of valvular interstitial cells and macrophage polarization. Methods and results In initial screening experiments, we investigated SOCS3-protein expression in explanted human aortic valves from patients undergoing surgical aortic valve replacement. SOCS3 is expressed in both stenotic and non-stenotic valve tissues. Immunofluorescence-staining of human aortic valves shows the co-localisation of SOCS3 with the interstitial cell-marker Vimentin and SOCS1, which is known to regulate macrophage-polarisation together with SOCS3 (Fig. 1). Staining of the macrophage-marker CD68 revealed its co-localisation with calcified areas of the aortic valve cusp. We validated our findings in our in vitro model of VIC calcification using two different calcifying conditions. Upregulation of RUNX2 and BMP2 verified successful osteogenic differentiation of VICs. During osteogenic differentiation for 7 days, SOCS3 and SOCS1 are significantly upregulated (Fig. 2A). We were able to induce EndMT by stimulating VEC with either TNFα or TGFβ/IL1β in vitro. We show that EndMT leads to the loss of endothelial cell markers like eNOS and VWF and to an upregulation of interstitial cell markers (such as Vimentin and α-SMA) and markers of EndMT (SNAI2). During EndMT, we were also able to observe a significant upregulation of SOCS1 and SOCS3 after 7 days (Fig. 2B). Ongoing knockdown experiments will help to elucidate the role of SOCS1 and SOCS3 during initiation and progression of CAVD. Conclusion We aim towards a better understanding of SOCS3 and its role in inflammation and calcification as hallmarks of the pathogenesis of CAVD. Since Zoledronic acid has been shown to induce a decreased expression of SOCS3 in macrophages, this might present as a possible treatment strategy to target SOCS3 therapeutically. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Deutsche Forschungsgemeinschaft

Enrichment in procoagulant microparticles in calcified human aortic valve – role in valvular endothelium alterations and enhanced thrombogenicity

Abstract Background Aortic stenosis (AS) is characterized by endothelial dysfunction (ED), inflammatory cell infiltration, myofibroblastic and osteoblastic differentiation. Subclinical leaflet thrombosis was recently linked to higher rates of stroke and transient ischemic attack after transcatheter aortic valve implantation (TAVI). Procoagulant microparticles (MPs) are associated with ED, inflammation and clot formation. There is limited evidence regarding intra-valvular MPs content and their potential biological effects. This question is particularly relevant in TAVI in which the residing native valve could constitute a source of thrombotic activity enhancing leaflet thrombosis and valve dysfunction. Purpose Therefore, we hypothesized that MPs trapped within the native aortic valve contribute to valvular dysfunction including enhanced thrombogenicity. Methods Human valves were collected from patients undergoing surgical valve replacement for AS or aortic insufficiency (AI). Pro-thrombotic, pro-inflammatory, and ED markers were identified in the calcified vs non-calcified part of the valves by Western-blot. Calcium content was measured through colorimetric method. MPs were extracted from human pathological valves, and quantified through their prothrombinase activity. Primary cultures of porcine valvular endothelial cells (VEC) were treated with the MPs (10 nmol/L) or thrombin (1U/ml) for 24hrs. Phenotypic change was appreciated through gene expression pattern assessed by RT-qPCR. IL-8 secretion was measured by ELISA. Results The phenotype of the AS valve was characterized through increased expression of thrombogenic (tissue factor, thrombomodulin, PAI-1), adhesive (VCAM-1, ICAM-1) and inflammatory (COX-1, COX-2) molecules in the calcified part of the valve. Moreover, MPs content was increased in the calcified vs non-calcified part of the valve or AI valves. MPs levels was correlated with valvular calcium content (R=0.3862: p&amp;lt;0.001). Tissue factor was increased in MPs extracted from AS vs AI. The biological effect of MPs was tested on VEC in-vitro. Results showed dramatic increase in expression of inflammatory cytokines (CXCL10, CCL11, CXCL8, MCP1) adhesion molecules (VCAM-1, ICAM-1, SELP, SELE) and proangiogenic factors (VEGFR2, ANGPTL4) in VEC exposed to MPs (24h) from AS vs AI. Enhanced secretory phenotype was evidenced through IL-8 determination in the supernatant of VEC stimulated with MPs from AS valve. Conclusion Calcified aortic valve is a potent reservoir of MPs, acting as a pro-thrombogenic source per se and promoting a switch of VEC phenotype toward prothrombotic, proinflammatory and proangiogenic pattern. These data suggest that MPs released from the native valve constitute an important source of mediators involved in enhanced thrombogenicity and valvular remodeling. Funding Acknowledgement Type of funding sources: Other. Main funding source(s): GERCA-Groupe Etudes Reali Commercia Avignon

LXR-beta signalling is a key mediator in the pathogenesis of aortic valve stenosis and its prevention by saringosterol

Abstract Background Cholesterol metabolism contributes as a risk factor for aortic valve stenosis (AS), but pharmacological approaches remained unsatisfying. The liver-X-receptor (LXR) is a key regulator in cholesterol metabolism, though its clinical use is limited due to unwanted side effects. The seaweed-derived oxysterol saringosterol is an agonist of the LXRβ, promising a more favourable tolerability. Purpose This study aimed to better understand the pathophysiology of aortic valve stenosis and to assess the potential of saringosterol as a targeted pharmacotherapy. Methods Tissue samples from aortic valves were collected from patients with AS or aortic valve regurgitation (AR). Transcriptomics were performed and gene ontology (GO) analysis was used to determine pathways and genes that are relevant to AS, and then validated using qPCR. In vivo, mice received a wire-induced aortic valve stenosis and were either fed a diet supplemented with saringosterol or control diet. Haemodynamic characteristics were assessed using echocardiography. Additionally, hepatic concentrations of saringosterol, expression of LXRβ regulated genes as well as aortic valve thickness and composition were assessed. In vitro, human aortic valve interstitial cells (VIC) were cultured in a procalcifing medium and stimulated with saringosterol to investigate the underlying molecular mechanisms. Results Transcriptomic analysis of AS samples revealed the regulation of several GO-terms related to cholesterol- or lipid metabolism. Many of the genes identified were regulated by LXRβ, suggesting its pathophysiological relevance in AS. We validated this assumption by performing qPCR from aortic valves for the most prominent downstream targets of LXRβ, ABCA1 and ABCG1, with both being differentially regulated. In vivo, treatment with saringosterol for six weeks resulted in a significant accumulation of saringosterol in liver tissue as well as induction of LXRβ-regulated genes. Furthermore, treatment with saringosterol strikingly reduced the development of AS after wire injury as assessed by echocardiographic and histological measurements. In vitro, the differentiation of VIC into osteoblastic and myofibroblastic phenotypes was abolished by saringosterol, which reduced the expression of the procalcifying mediators RUNX-2 and ACTA-2 in a dose-dependent manner. Conclusion We identified LXRβ-signalling as a key regulator in the pathophysiology of AS. Transcriptomic analyses revealed that cholesterol metabolism was altered in human AS, and many of the genes involved were linked to the LXRβ. In a murine model, we demonstrated that oral application of saringosterol induced LXRβ-activity and mitigated the development of AS. In vitro experiments demonstrated that saringosterol prevents adverse cell differentiation of VIC, which provides a mechanistic explanation. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): BONFOR Universität Bonn

Modulation of inflammatory M1-macrophages phenotype by valvular interstitial cells

BackgroundAortic valve stenosis involves inflammation, excess deposition of a collagen-rich extracellular matrix, and calcification. Recent studies have shown that M1 or inflammatory macrophages derived from infiltrating monocytes promote calcification of valvular interstitial cells, the most prevalent cell type of the aortic valve. We hypothesized that valvular interstitial cells could modulate inflammatory macrophages phenotype. MethodsWe first assessed macrophage phenotype in human aortic valve stenosis and control aortic valves from donors. Then, we examined profibrotic and inflammatory-related gene expression in valves and valvular interstitial cells. Finally, we investigated whether valvular interstitial cells can modify the phenotype of inflammatory macrophages. ResultsCirculating monocytes and plasma transforming growth factor beta-1 levels of patients with aortic valve stenosis were significantly higher compared with patients without aortic valve stenosis. Histologic analysis of thickened spongiosa of the aortic valve from patients with aortic valve stenosis showed a high macrophage infiltration but a low matrix metalloproteinase-9 expression compared with control aortic valves. On the other hand, valvular interstitial cell culture of aortic valve stenosis exhibited a profibrotic phenotype with a high expression of transforming growth factor beta-1 and transforming growth factor beta-1/transforming growth factor beta-3 ratio but a decreased expression of the peroxisome proliferator-activated receptor gamma nuclear receptor. Valvular interstitial cell–conditioned media of aortic valve stenosis led to a decrease in enzymatic activity of matrix metalloproteinase-9 and an increase in production of collagen in inflammatory macrophages compared with valvular interstitial cell–conditioned media from control aortic valve donors. ConclusionsThese findings indicate that profibrotic valvular interstitial cells promote the imbalance of extracellular matrix remodeling by reducing matrix metalloproteinase-9 production on inflammatory macrophages that lead to excessive collagen deposition observed in aortic valve stenosis. Further investigation is needed to clarify the role of transforming growth factor beta-1/proliferator-activated receptor gamma nuclear receptor/matrix metalloproteinase-9 in aortic valve stenosis.