Calcific Aortic Valve Disease Research Articles

Progress on long non-coding RNAs in calcific aortic valve disease

Calcific aortic valve disease (CAVD) is a common cardiovascular condition in the elderly population. The aortic valve, influenced by factors such as endothelial dysfunction, inflammation, oxidative stress, lipid metabolism disorders, calcium deposition, and extracellular matrix remodeling, undergoes fibrosis and calcification, ultimately leading to stenosis. In recent years, long non-coding RNAs (lncRNAs) have emerged as significant regulators of gene expression, playing crucial roles in the occurrence and progression of various diseases. Research has shown that lncRNAs participate in the pathological process underlying CAVD by regulating osteogenic differentiation and inflammatory response of valve interstitial cells. Specifically, lncRNAs, such as H19, MALAT1, and TUG1, are closely associated with CAVD. Some lncRNAs can act as miRNA sponges, form complex regulatory networks, and modulate the expression of calcification-related genes. In brief, this review discusses the mechanisms and potential therapeutic targets of lncRNAs in CAVD.

Similar, but not the same: multiomics comparison of human valve interstitial cells and osteoblast osteogenic differentiation expanded with an estimation of data-dependent and data-independent PASEF proteomics

Abstract Osteogenic differentiation is crucial in normal bone formation and pathological calcification, such as calcific aortic valve disease (CAVD). Understanding the proteomic and transcriptomic landscapes underlying this differentiation can unveil potential therapeutic targets for CAVD. In this study, we employed RNA sequencing transcriptomics and proteomics on a timsTOF Pro platform to explore the multiomics profiles of valve interstitial cells (VICs) and osteoblasts during osteogenic differentiation. For proteomics, we utilized 3 data acquisition/analysis techniques: data-dependent acquisition (DDA)–parallel accumulation serial fragmentation (PASEF) and data-independent acquisition (DIA)–PASEF with a classic library-based (DIA) and machine learning–based library-free search (DIA-ML). Using RNA sequencing data as a biological reference, we compared these 3 analytical techniques in the context of actual biological experiments. We use this comprehensive dataset to reveal distinct proteomic and transcriptomic profiles between VICs and osteoblasts, highlighting specific biological processes in their osteogenic differentiation pathways. The study identified potential therapeutic targets specific for VICs osteogenic differentiation in CAVD, including the MAOA and ERK1/2 pathway. From a technical perspective, we found that DIA-based methods demonstrate even higher superiority against DDA for more sophisticated human primary cell cultures than it was shown before on HeLa samples. While the classic library-based DIA approach has proved to be a gold standard for shotgun proteomics research, the DIA-ML offers significant advantages with a relatively minor compromise in data reliability, making it the method of choice for routine proteomics.

Iron Deficiency Promotes Intra-leaflet Haemorrhage-induced Aortic Valve Calcification: an Experimental Study.

Interleaflet haemorrhage (IH) plays a well-recognized detrimental role in calcified aortic valve disease (CAVD). However, IH-induced fibro-osteogenic responses in valvular interstitial cells (VICs) appear to be triggered under specific pathological conditions. Iron deficiency (ID), a common co-morbidity in CAVD, may influence these responses. This study investigated the relationship between ID and pathological changes associated with CAVD, and its effects on IH-mediated fibro-osteogenic differentiation of VICs. Two independent studies were conducted, including 2,495 patients in the discovery study and 34 in the validation study. Our data demonstrated that ID was associated with CAVD severity and progression, particularly in an age-dependent manner. Based on these clinical findings, immunofluorescence and Western blot analyses revealed that TFR1, a key iron import transporter, was significantly upregulated in human calcified aortic valves. Concurrently, iron accumulation was detected by Perl's staining in both calcific and non-calcific valve sections. In vitro, VICs cultured with human serum from ID patients showed red blood cell lysis-induced iron overload and fibro-calcific differentiation. ID triggers TFR1-mediated intracellular iron overload, leading to fibrosis and calcification in human VICs, thereby contributing to IH-mediated valve remodeling and calcification. These findings supported the potential role of monitoring and correcting ID to slow or prevent the progression of valvular calcification.

Research progress on the interaction between valve endothelial cells and interstitial cells in calcific aortic valve disease

Research progress on the interaction between valve endothelial cells and interstitial cells in calcific aortic valve disease

The association between triglyceride to high-density-lipoprotein cholesterol ratio and calcific aortic valve disease: a retrospective study

BackgroundThe ratio of triglycerides to high-density-lipoprotein cholesterol (TG/HDL-C) is increasingly recognized as a practical marker for insulin resistance and cardiovascular risk assessment. This retrospective study investigates the potential of the TG/HDL-C ratio to predict the development of calcific aortic valve disease (CAVD), thereby extending its applicability in cardiovascular diagnostics.MethodsData from 400 individuals, comprising 200 patients with diagnosed CAVD and 200 matched healthy controls, were analyzed. Clinical parameters were compared between groups, and logistic regression was utilized to explore the association of the TG/HDL-C ratio with CAVD. The diagnostic performance of the TG/HDL-C ratio was assessed using receiver operating characteristic (ROC) curves.ResultsThe TG/HDL-C ratio was notably higher in the CAVD group than in the controls (Z = -7.98, P < 0.001). Multivariable logistic regression analysis indicated that the TG/HDL-C ratio is an independent predictor of CAVD after adjusting for confounders including gender. The ROC curve analysis revealed that the TG/HDL-C ratio achieved a sensitivity of 80.5%, a specificity of 59.5%, and an area under the curve (AUC) of 0.731 (P < 0.001), confirming its efficacy in predicting CAVD.ConclusionsHigh TG/HDL-C ratio was significantly associated with the occurrence of CAVD, and the TG/HDL-C ratio could be used as a potential diagnostic tool and risk assessment indicator for CAVD.Clinical trial numberNot applicable.

Side- and Disease-Dependent Changes in Human Aortic Valve Cell Population and Transcriptomic Heterogeneity Determined by Single-Cell RNA Sequencing.

Calcific aortic valve disease (CAVD) is a highly prevalent disease, especially in the elderly population, but there are no effective drug therapies other than aortic valve repair or replacement. CAVD develops preferentially on the fibrosa side, while the ventricularis side remains relatively spared through unknown mechanisms. We hypothesized that the fibrosa is prone to the disease due to side-dependent differences in transcriptomic patterns and cell phenotypes. To test this hypothesis, we performed single-cell RNA sequencing using a new method to collect endothelial-enriched samples independently from the fibrosa and ventricularis sides of freshly obtained human aortic valve leaflets from five donors, ranging from non-diseased to fibrocalcific stages. From the 82,356 aortic valve cells analyzed, we found 27 cell clusters, including seven valvular endothelial cell (VEC), nine valvular interstitial cell (VIC), and seven immune, three transitional, and one stromal cell population. We identified several side-dependent VEC subtypes with unique gene expression patterns. Homeostatic VIC clusters were abundant in non-diseased tissues, while VICs enriched with fibrocalcific genes and pathways were more prevalent in diseased leaflets. Furthermore, homeostatic macrophage (MΦ) clusters decreased while inflammatory MΦ and T-cell clusters increased with disease progression. A foamy MΦ cluster was increased in the fibrosa of mildly diseased tissues. Some side-dependent VEC clusters represented non-diseased, protective phenotypes, while others were CAVD-associated and were characterized by genes enriched in pathways of inflammation, endothelial-mesenchymal transition, apoptosis, proliferation, and fibrosis. Interestingly, we found several activator protein-1 (AP-1)-related transcription factors (FOSB, FOS, JUN, JUNB) and EGR1 to be upregulated in the fibrosa and diseased aortic valve leaflets. Our results showed that VECs are highly heterogeneous in a side- and CAVD-dependent manner. Unique VEC clusters and their differentially regulated genes and pathways found in the fibrosa of diseased tissues may represent novel pathogenic mechanisms and potential therapeutic targets.

Retraction: MicroRNA-34c inhibits osteogenic differentiation and valvular interstitial cell calcification via STC1-mediated JNK pathway in calcific aortic valve disease.

[This retracts the article DOI: 10.3389/fphys.2020.00829.].

Global, Regional, and National Burden of Valvular Heart Disease, 1990 to 2021.

Valvular heart disease poses an escalating global health challenge with an increasing impact on mortality and disability. This study aims to comprehensively analyze the global burden of valvular heart disease. Using the Global Burden of Disease 2021 data, we analyzed the prevalence and disability-adjusted life years, examining implications across demographics and geographic regions. In 2021, an estimated 54.8 million (95% uncertainty interval [UI], 43.3-67.6) cases of rheumatic heart disease, 13.3 million (95% UI, 11.4-15.2) cases of nonrheumatic calcific aortic valve disease (CAVD), and 15.5 million (95% UI, 14.5-16.7) cases of nonrheumatic degenerative mitral valve disease (DMVD) were reported globally. Despite the rising prevalence, disability-adjusted life years declined between 1991 and 2021. Among individuals aged 70 years or older, the age-standardized prevalences were 1803.6 per 100 000 (95% UI, 1535.5-2055.7) for CAVD and 2148.9 per 100 000 (95% UI, 2001.4-2310.1) for DMVD. Sub-Saharan Africa had the highest age-standardized prevalence for rheumatic heart disease; Conversely, high-income regions led in CAVD and DMVD prevalence. Rheumatic heart disease had the highest age-standardized prevalence of 1184.2 per 100 000 (95% UI, 932.4-1478.2) in low Socio-Demographic Index (SDI) regions, whereas CAVD peaked at 349.8 per 100 000 (95% UI, 303.6-395.8) in high SDI regions. The most substantial increases in age-standardized prevalences of CAVD from 1990 to 2021 occurred in the middle SDI and low-middle SDI regions. A parallel trend was noted for DMVD. Rheumatic heart disease remains a significant burden in low SDI regions, whereas CAVD and DMVD pose challenges in high SDI regions with aging populations.

The m6A demethylase ALKBH5 is a novel epigenetic regulator of aortic valve calcification.

Calcific aortic valve disease (CAVD) is a common heart valve disease with significant clinical consequences. The mechanisms that drive the pathogenesis of CAVD remain to be fully elucidated. N6-methyladenosine (m6A), the most prevalent RNA epigenetic regulator, has recently been implicated in cardiovascular disease, but its role in CAVD has yet to be investigated. In this study, we investigated the potential function of m6A modification in CAVD. Using clinical samples from CAVD patients in combination with human valve interstitial cell (hVIC) calcification model, we screened the expression of m6A modulators and discovered that ALKBH5 alkB homolog 5, RNA demethylase (ALKBH5), a key m6A demethylase, was significantly down-regulated in calcified hVICs and human aortic valves. Consistently, increased m6A levels were seen in calcified hVICs, and treated with 3-deazaadenosine (DAA), an inhibitor of m6A modification, significantly reduced hVIC osteogenic differentiation and calcification. In addition, we showed that silencing of ALKBH5 expression increased global m6A levels, and accelerated hVIC osteogenic differentiation and calcification, whereas overexpression of ALKBH5 resulted in the opposite effect. We demonstrated that ALKBH5 directly modulate m6A levels of TGFBR2 and its mRNA stability, leading to altered TGFBR2 expression and SMAD2 signaling in hVICs. We further showed that inhibition of TGFBR2 or knockdown of SMAD2 attenuated ALKBH5 knockdown-induced hVIC osteogenic differentiation and calcification. The expression of the m6A reader protein YTH N6-methyladenosine RNA binding protein F1 (YTHDF1) was upregulated during the process of hVIC calcification. Intriguingly, we revealed that the ALKBH5 silencing-induced increased hVIC osteogenic differentiation and calcification were abolished after knockdown of YTHDF1. These data suggest a potential role YTHDF1 in aortic valve calcification. This study showed that ALKBH5 attenuated aortic valve calcification through the TGFBR2/SMAD2 signaling pathway via direct m6A modification of TGFBR2. .

Calcific aortic stenosis: omics-based target discovery and therapy development.

Calcific aortic valve disease (CAVD) resulting in aortic stenosis (AS) is the most common form of valvular heart disease, affecting 2% of those over age 65. Those who develop symptomatic severe AS have an average further lifespan of <2 years without valve replacement, and three-quarters of these patients will develop heart failure, undergo valve replacement, or die within 5 years. There are no approved pharmaceutical therapies for AS, due primarily to a limited understanding of the molecular mechanisms that direct CAVD progression in the complex haemodynamic environment. Here, advances in efforts to understand the pathogenesis of CAVD and to identify putative drug targets derived from recent multi-omics studies [including (epi)genomics, transcriptomics, proteomics, and metabolomics] of blood and valvular tissues are reviewed. The recent explosion of single-cell omics-based studies in CAVD and the pathobiological and potential drug discovery insights gained from the application of omics to this disease area are a primary focus. Lastly, the translation of knowledge gained in valvular pathobiology into clinical therapies is addressed, with a particular emphasis on treatment regimens that consider sex-specific, renal, and lipid-mediated contributors to CAVD, and ongoing Phase I/II/III trials aimed at the prevention/treatment of AS are described.

Macrophages in Calcific Aortic Valve Disease: Paracrine and Juxtacrine Disease Drivers.

A significant role in the pathogenesis of CAVD is played by innate immunity cells, such as macrophages. In stenotic valves, macrophages have enhanced inflammatory activity, and the population's balance is shifted toward pro-inflammatory ones. Pro-inflammatory macrophages release cytokines, chemokines, and microRNA, which can directly affect the resident valvular cells and cause valve calcification. In CAVD patients, macrophages may have more pronounced pro-inflammatory properties, enhanced not only by paracrine signals but also by juxtacrine Notch signaling and epigenetic factors, which influence the maturation of macrophages' progenitors. In this review, we observe the accumulated data on the involvement of macrophages in CAVD development via paracrine and juxtacrine interactions.

Oxidized Phospholipids and Calcific Aortic Valvular Disease

BackgroundOxidized phospholipids (OxPLs) are carried by apolipoprotein B-100–containing lipoproteins (OxPL-apoB) including lipoprotein(a) (Lp[a]). Both OxPL-apoB and Lp(a) have been associated with calcific aortic valve disease (CAVD). ObjectivesThis study aimed to evaluate the associations between OxPL-apoB, Lp(a) and the prevalence, incidence, and progression of CAVD. MethodsOxPL-apoB and Lp(a) were evaluated in MESA (Multi-Ethnic Study of Atherosclerosis) and a participant-level meta-analysis of 4 randomized trials of participants with established aortic stenosis (AS). In MESA, the association of OxPL-apoB and Lp(a) with aortic valve calcium (AVC) at baseline and 9.5 years was evaluated using multivariable ordinal regression models. In the meta-analysis, the association between OxPL-apoB and Lp(a) with AS progression (annualized change in peak aortic valve jet velocity) was evaluated using multivariable linear regression models. ResultsIn MESA, both OxPL-apoB and Lp(a) were associated with prevalent AVC (OR per SD: 1.19 [95% CI: 1.07-1.32] and 1.13 [95% CI: 1.01-1.27], respectively) with a significant interaction between the two (P < 0.01). Both OxPL-apoB and Lp(a) were associated with incident AVC at 9.5 years when evaluated individually (interaction P < 0.01). The OxPL-apoB∗Lp(a) interaction demonstrated higher odds of prevalent and incident AVC for OxPL-apoB with increasing Lp(a) levels. In the meta-analysis, when analyzed separately, both OxPL-apoB and Lp(a) were associated with faster increase in peak aortic valve jet velocity, but when evaluated together, only OxPL-apoB remained significant (ß: 0.07; 95% CI: 0.01-0.12). ConclusionsOxPL-apoB is a predictor of the presence, incidence, and progression of AVC and established AS, particularly in the setting of elevated Lp(a) levels, and may represent a novel therapeutic target for CAVD.

Abstract 4136901: Mechanosensitive channel protein Piezo1 synergizes with TLR4 to enhance osteogenic responses in human aortic valve interstitial cells

Background: Calcific aortic valve disease (CAVD), a common heart valve disease in the elderly, has a complex pathogenesis. Abnormal blood flow shear can be regarded as an important factor in the development of CAVD, and a key molecule that responds to mechanical stress is the mechanosensitive ion channel protein Piezo1; however, the role of Piezo1 in CAVD has not been clarified. Methods and results: We collected CAVD patients and their age- and sex-matched controls, and compared the differences in the protein expression levels of Piezo1 and TLR4 between the two using Western blot and other techniques, and found that the expression of Piezo1 and TLR4 proteins was elevated in the CAVD group. We gave LPS or Piezo1 agonist Yoda1 to stimulate aortic valve mesenchymal stromal cells, respectively, and the Western blot results showed that ALP and Runx2 expression were elevated after LPS and Yoda1 stimulation, and immunofluorescence and alizarin red staining showed similar results. Inhibition of TLR4 or Piezo1 attenuated the osteogenic differentiation of AVICs. Cellular immunofluorescence results showed that TLR4 could be transported from the Golgi to the cell membrane under Yoda stimulation, and Golgi-cell membrane transport of TLR4 was attenuated after Rab3b knockdown by siRNA. After first pre-stimulation with Yoda1 and then stimulation with LPS, Western blot and immunofluorescence results showed that the protein levels of ALP and Runx2 as well as NF-κB phosphorylation were to be significantly increased. Conclusion: Stimulation of Piezo1 and TLR4 can enhance the osteogenic response of AVICs. Stimulation of Piezo1 enhances the transport of TLR4 from the Golgi to the cell membrane via Rab3b, and Piezo1 synergistically enhances the osteogenic response of AVICs with TLR4, thus promoting the development of CAVD.

Abstract 4138340: Cav-1/FGF2 Axis Mediates Endothelial-Mesenchymal Transition Promoting Aortic Valve Calcification

Background: Calcific Aortic Valve Disease is a common cardiac condition with no effective medication available; severe cases require surgery. Endothelial-to-mesenchymal transition is a key pathological process, yet the genetic regulatory mechanisms involved remain unclear. Research Questions: Investigating the mechanism of Cav-1/FGF2 axis-mediated endothelial-mesenchymal transition in aortic valve calcification. Methods: Histopathology, immunohistochemical staining, and western blot analysis were performed on 5 non-calcified and 5 calcified aortic valves. Proteomic sequencing was conducted on these samples. Single-cell RNA sequencing on 3 calcified aortic valves focused on valve endothelial and interstitial cells to explore key genes and functions in EndoMT. In vivo and in vitro experiments, along with single-cell multi-omics sequencing, elucidated the role and mechanism of the Cav-1/FGF2 axis in EndoMT using endothelial cell-specific EndoMT knockout mouse models and cell models. Results: Proteomics and single-cell sequencing results indicated that Cav-1 is involved in the Endothelial-to-mesenchymal transition process in Calcific Aortic Valve Disease. Cav-1 plays a crucial role in the intermediate transition of EndoMT, interacting significantly with FGF2. Immunohistochemical staining showed high expression of Cav-1 in calcified aortic valves. In vitro experiments demonstrated that Cav-1 mediates Endothelial-to-mesenchymal transition in valve endothelial cells, promoting aortic valve calcification. Molecular docking analysis revealed potential direct interaction between Cav-1 and FGF2 proteins. Elevated Cav-1 gene expression increases the number of cell surface caveolae, enhancing cell surface area, which promotes FGF2 binding to its receptor and activates the TGF-beta receptor, both of which facilitate EndoMT through the Smad pathway. Conclusion(s): Activation of the Cav-1/FGF2 axis promotes endothelial-mesenchymal transition, thereby promoting aortic valve calcification.

Abstract 4119560: Oxidized Phospholipids and Calcific Aortic Valvular Disease

Introduction: Oxidized phospholipids (OxPL) are carried by apolipoprotein B-100-containing lipoproteins (OxPL-apoB) including lipoprotein(a) [Lp(a)]. Both OxPL-apoB and Lp(a) have been associated with calcific aortic valve disease (CAVD). Aims: We aimed to evaluate the independent associations between OxPL-apoB, Lp(a) and the prevalence, incidence, and progression of CAVD. Methods: OxPL-apoB and Lp(a) were evaluated in the Multi-Ethnic Study of Atherosclerosis (MESA) and a participant-level meta-analysis of four randomized trials of participants with established aortic stenosis (AS). In MESA, the association of OxPL-apoB and Lp(a) with aortic valve calcium (AVC) at baseline and 9.5 years was evaluated using multivariable ordinal regression models. In the meta-analysis, the association between OxPL-apoB and Lp(a) with AS progression (annualized change in peak aortic valve jet velocity (V max )) was evaluated using multivariable linear regression models. Results: In MESA, both OxPL-apoB and Lp(a) were independently associated with prevalent AVC (OR (95% CI) per SD: 1.19 (1.07-1.32) and 1.13 (1.01-1.27), respectively) with a significant interaction between the two (p&lt;0.01). Both OxPL-apoB and Lp(a) were associated with incident AVC at 9.5 years when evaluated independently (interaction p&lt;0.01). The OxPL-apoB*Lp(a) interaction demonstrated higher odds of prevalent and incident AVC for OxPL-apoB with increasing Lp(a) levels. Mediation analyses demonstrated that Lp(a) partially mediated the association between OxPL-apoB and AVC. In a 2x2 analysis, the greatest risk for both prevalent and incident AVC was observed when Lp(a) and OxPL-apoB were both above the 80 th percentile ( Figure ). In the meta-analysis, when analyzed separately, both OxPL-apoB and Lp(a) were independently associated with faster increase in V max , but when evaluated together, only OxPL-apoB remained significant (ß 0.07, 95% CI 0.01-0.12, Figure ). Conclusions: OxPL-apoB is an independent predictor of the presence, incidence and progression of AVC and established AS, particularly in the setting of elevated Lp(a) levels and may represent a novel therapeutic target for CAVD.

Niclosamide attenuates calcification in human heart valvular interstitial cells through inhibition of the AMPK/mTOR signaling pathway

Calcific aortic valve disease (CAVD) is a considerable health burden with a lack of effective therapeutic options. There is an urgent need to develop interventions that inhibit the osteogenic transformation of valvular interstitial cells (VICs) and delay the calcification process. Niclosamide, an FDA-approved anti-helminthic drug, has emerged as a promising candidate that demonstrates a negative regulatory effect on porcine VICs calcification. However, its molecular mechanism in human VICs (hVICs) remains to be investigated. In this study, high-resolution mass spectrometry-based proteomics and phosphoproteomics were employed, and 8373 proteins and 3697 phosphosites were identified in hVICs treated with a pro-calcifying medium and niclosamide. The quantitative proteomic and phosphoproteomic analysis resulted in the identification of calcification markers and osteogenesis-associated proteins. Bioinformatic analysis of the protein–protein interaction network and affected kinase prediction revealed that the AMPK/mTOR/p70S6K signaling cascade was altered upon calcific induction and niclosamide treatment. Further validation indicated that niclosamide inhibited the calcification of hVICs by targeting the mammalian target of the rapamycin (mTOR) signaling pathway. This study provides the first evidence that niclosamide could prevent osteoblastic differentiation in hVICs partially through the inhibition of the AMPK/mTOR/p70S6k signaling pathway, thereby mitigating hVICs calcification. These findings present a foundation for potential therapeutic strategies to impede the progression of CAVD and provide valuable insights into the pharmacological effects of niclosamide on human VICs.

Ligustrazine alleviates the progression of coronary artery calcification by inhibiting caspase-3/GSDME mediated pyroptosis.

Coronary artery calcification (CAC) is an early marker for atherosclerosis and is mainly induced by the osteoblast-like phenotype conversion of vascular smooth muscle cells (VSMCs). Recent reports indicate that NOD-like receptor protein 3 (NLRP3)-mediated pyroptosis plays a significant role in the calcification of vascular smooth muscle cells (VSMCs), making it a promising target for treating calcific aortic valve disease (CAC). Ligustrazine, or tetramethylpyrazine (TMP), has been found effective in various cardiovascular and cerebrovascular diseases and is suggested to inhibit NLRP3-mediated pyroptosis. However, the function of TMP in CAC is unknown. Herein, influences of TMP on β-glycerophosphate (β-GP)-stimulated VSMCs and OPG-/- mice were explored. Mouse Aortic Vascular Smooth Muscle (MOVAS-1) cells were stimulated by β-GP with si- caspase-3, si- Gasdermin E (GSDME) or TMP. Increased calcification, reactive oxygen species (ROS) level, Interleukin-1beta (IL-1β) and Interleukin-18 (IL-18) levels, lactate dehydrogenase (LDH) release, enhanced apoptosis, and activated cysteine-aspartic acid protease-3 (caspase-3)/GSDME signaling were observed in β-GP-stimulated MOVAS-1 cells, which was sharply alleviated by si-caspase-3, si-GSDME or TMP. Furthermore, the impact of TMP on the β-GP-induced calcification and injury in MOVAS-1 cells was abolished by raptinal, an activator of caspase-3. Subsequently, OPG-/- mice were dosed with TMP or TMP combined with raptinal. Calcium deposition, increased nodules, elevated IL-1β and IL-18 levels, upregulated CASP3 and actin alpha 2, smooth muscle (ACTA2), and activated caspase-3/GSDME signaling in OPG-/- mice were markedly alleviated by TMP, which were notably reversed by the co-administration of raptinal. Collectively, TMP mitigated CAC by inhibiting caspase-3/GSDME mediated pyroptosis.

Targeting an altered sphingolipid profile with consecutive lipotoxicity as therapeutic approach to calcific aortic valve disease

Abstract Background Aortic stenosis due to calcific aortic valve disease (CAVD) is the most common valvular heart disease. In symptomatic patients, without repair the prognosis is poor. Treatment to date is based on surgery or catheter-based interventions. Pharmacological therapy options to prevent the progression of valve calcification are not available. In vascular smooth muscle cells, cellular accumulation of sphingolipids is associated with a proinflammatory response in the sense of lipotoxicity. Chronic inflammatory processes are indeed also essential for aortic valve calcification. Purpose With the subsequent goal of developing innovative treatment approaches for CAVD, we therefore aim to elucidate the unclear role of sphingolipids in the development of CAVD. Methods Human aortic valve and plasma samples of CAVD patients and controls (n=26) were analysed by liquid chromatography–mass spectrometry for lipidomics and proteomics. Human aortic valve interstitial cells (hVICs) were stimulated with ceramides. Calcification was detected by alizarin red staining. mRNA expression of bone-related proteins was determined by qPCR. NF-kB pathway was assessed by proteome profiler. Myriocin and GW4869 were applied to inhibit ceramide biosynthesis. PDTC and MCC950 were used to inhibit NF-kB Pathway and NLRP3 activation. ApoE-/- mice received a gain-of-function PCSK9 adeno-associated virus vector and fed high cholesterol diet for 14 weeks. Myriocin was applied orally over the treatment period to inhibit ceramide de novo synthesis in vivo. Murine echocardiography was used to measure transvalvular velocity and left ventricular function. Calcification degree of murine aortic valve was determined by histological staining. Results A significantly altered sphingolipid profile was observed in aortic valve tissue of CAVD patients (Fig. 1). Ceramide species e.g. Cer (17:1;2O/16:0) were increased. Verifying the biological relevance of these findings in vitro, C2-Cer (d18:1/2:0) enhanced hVIC calcification (Fig. 2). Accordingly, ALP activity and mRNA expression of osteogenic genes RUNX2, ALPL and MSX2 were increased. Furthermore, C2-Cer (d18:1/2:0) enhanced NF-kB p65 phosphorylation and NLRP3 activation, while treatment with both PDTC and MCC950 protected against C2-Cer (d18:1/2:0) induced calcification. Supporting the role of sphingolipid biosynthesis in hVIC calcification, Myriocin and GW4869 both reduced ox-LDL-induced VIC calcification which was reversed by C2-Cer(d18:1/2:0). Finally, Myriocin reduced the degree of calcification and transvalvular jet velocity of aortic valve in the PCSK9-AAV ApoE-/- mice model. Conclusion CAVD is accompanied by an accumulation of ceramides causing lipotoxicity in human aortic valve tissue. Pharmacological modulation of sphingolipid metabolism is an effective approach to prevent the development and progression of aortic valve calcification in vivo and should be considered as a future therapeutic strategy in CAVD patients.Volcano Plot of CAVD tissue lipidomicsC2 Ceramide enhances hVIC calcification

Cardiac damage predicts the outcome of calcific aortic valve disease regardless of the severity of aortic stenosis

Abstract Title: Cardiac damage predicts the outcome of patients with calcific aortic valve disease in both mild and moderate aortic stenosis Background Although the burden of calcific aortic valve disease (CAVD) is increasing, there is no current universal consensus for prognostic prediction. A staging system based on cardiac damage has been proposed to better stratify risk and evaluate the benefit of aortic valve intervention (AVI). Purpose We sought to evaluate this staging system’s prognostic value in mild and moderate aortic stenosis (AS). Methods Data from clinically indicated echocardiograms performed between 2010 and 2018 in patients aged over 18 years were extracted and linked to national outcome data. A time to first event analysis was performed with a primary outcome of mortality or heart failure hospitalization. Stages were categorized as described in previous literature: no extravalvular damage (stage 0); left ventricular damage (stage 1); left atrial damage (stage 2); elevated right ventricular pressures (stage 3); or right ventricular damage (stage 4). Results Amongst 24,699 patients in the study cohort, 513 had moderate AS and 920 had mild AS. Associated cardiac damage in those with moderate AS was graded as follows: Stage 0 - 9.4%; Stage 1 - 53.7%; Stage 2 - 31.1%; Stage 3 - 3.2%; Stage 4 - 2.6%. Increasing severity of CAVD correlated closely with increased risk of the primary outcome in both moderate (HR 1.62 per stage; 95% CI 1.36 – 1.92) and mild AS (HR 1.93 per stage; 95% CI 1.69 – 2.21), whilst increasing stage of cardiac damage was associated with mortality when censoring at the time of AVI in both moderate (HR 1.97 per stage; 95% CI 1.66 – 2.34) and mild AS (HR 2.06 per stage; 95% CI 1.81 – 2.35). Conclusion Cardiac dysfunction predicts prognosis to a similar extent in both mild and moderate AS. These data suggest that adverse outcomes may not be fully attributable to the haemodynamic consequences of valve disease, and therefore may not be reversed by valve intervention. Strategies to treat cardiac dysfunction in parallel with management of the anatomical valve pathology may be required.Mortality in mild AS by CAVD stageMortality in moderate AS by CAVD stage

Paired single-cell RNA sequencing and T cell receptor sequencing reveals both pro- and anti-inflammatory roles of T cells in patients with calcific aortic valve disease

Abstract Background Calcified aortic valve disease (CAVD) is a chronic unresolvable inflammatory disorder that affects 25% of adults over 65 years old, leading to aortic stenosis, heart failure, and death [1]. Since the molecular mechanisms of its pathogenesis are not well understood, therapies to treat root causes of the disease remain to be developed, with valve replacement as the major intervention strategy currently. Increasing evidence indicates that substantial T cells infiltrate in the aortic valve during calcification, and clonal expanded CD8+ T cells have been identified in the calcificed aortic valve by bulk T cell receptor (TCR) repertoire sequencing [2]. Purpose To decipher T cell heterogenity and understand the precise mechanisms of T cell subsets involving CAVD progression as a basis for novel therapeutic targets. Methods In Cohort 1, we collected aortic valves from CAVD (n=3) and healthy (n=2) patients for single-cell RNA sequencing (scRNA-seq). In Cohort 2, we performed paired scRNA-seq and TCR sequencing (scTCR-seq) on aortic valves obtained from CAVD (n=3) and healthy (n=3) patients to evaluate both gene expression and clonality. Pathway enrichment analysis and cell-cell interaction analysis elucidated the function of T cells in the calcificied valves. Immunohistochemistry and fluorescence-activated cell sorting were recruited to verify the key findings. Results scRNA-seq demonstrated a conversion from anti-inflammatory regulatory T (Treg) cells towards pro-inflammatory T helper 17 (Th17) cells in the calcificied aortic valve. Paired scRNA-seq and scTCR-seq revealed CAVD-related signaling pathways (osteoclast differentiation, NOD-like receptor signaling pathways, IL-17 signaling pathways) significantly upregulated in highly expanded CD8+ T cells in the calcified valve, with upregulation of pro-inflammatory mediators such as IFNG, CCL4 and CCL5, suggesting pro-inflammation functions in clonally expanded T cells. Moreover, we characterized tissue-resident memory T cells, which resembled similar transcriptome and TCR clonality as exhausted T cells in calcified valves. These cells up-regulated anti-inflammation-related transcriptomes but their cellular fraction decreased in calcified valves, implying anti-inflammatory functions of these tissue-resident memory and exhausted T cells. Conclusion This study elucidates transcriptomic and immune profiling of T cells in the calcified aortic valve, therefore shedding light on the pathophysiological mechanisms underlying aortic valve calcification from the novel perspective of tissue-specific T lymphocytes, providing promising targets for immune checkpoint-based therapeutic interventions.