Progression Of Aortic Valve Calcification Research Articles

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

Current Management and Therapy of Severe Aortic Stenosis and Future Perspective.

Intervention for severe aortic stenosis (AS) has dramatically progressed since the introduction of transcatheter aortic valve replacement (TAVR). Decades ago, controversies existed regarding comparing clinical outcomes between TAVR and surgical aortic valve replacement (SAVR) in various risk profiles. Recently, we discussed the durability of transcatheter heart valves and their lifetime management after aortic valve replacement (AVR). Regarding the management of AS, we discuss the appropriate timing of intervention for severe aortic stenosis, especially in asymptomatic patients. In spite of dramatic progression of intervention for AS, there are no established medications available to prevent or slow the progression of AS at present. Basic research and genome studies have suggested several targets associated with the progression of aortic valve calcification. Randomized controlled trials evaluating the efficacy of medications to prevent AS progression are ongoing, which might lead to new strategies for AS management. In this review, we summarize the current management of AS and the drugs expected to prevent the progression of AS.

AVCAPIR: A Novel Procalcific PIWI-Interacting RNA in Calcific Aortic Valve Disease.

Calcification of the aortic valve leads to increased leaflet stiffness and consequently results in the development of calcific aortic valve disease (CAVD). However, the underlying molecular and cellular mechanisms of calcification remain unclear. Here, we identified a novel aortic valve calcification-associated PIWI-interacting RNA (piRNA; AVCAPIR) that increases valvular calcification and promotes CAVD progression. Using piRNA sequencing, we identified piRNAs contributing to the pathogenesis of CAVD that we termed AVCAPIRs. High-cholesterol diet-fed ApoE-/- mice with AVCAPIR knockout were used to examine the role of AVCAPIR in aortic valve calcification (AVC). Gain- and loss-of-function assays were conducted to determine the role of AVCAPIR in the induced osteogenic differentiation of human valvular interstitial cells. To dissect the mechanisms underlying AVCAPIR-elicited procalcific effects, we performed various analyses, including an RNA pulldown assay followed by liquid chromatography-tandem mass spectrometry, methylated RNA immunoprecipitation sequencing, and RNA sequencing. RNA pulldown and RNA immunoprecipitation assays were used to study piRNA interactions with proteins. We found that AVCAPIR was significantly upregulated during AVC and exhibited potential diagnostic value for CAVD. AVCAPIR deletion markedly ameliorated AVC in high-cholesterol diet-fed ApoE-/- mice, as shown by reduced thickness and calcium deposition in the aortic valve leaflets, improved echocardiographic parameters (decreased peak transvalvular jet velocity and mean transvalvular pressure gradient, as well as increased aortic valve area), and diminished levels of osteogenic markers (Runx2 and Osterix) in aortic valves. These results were confirmed in osteogenic medium-induced human valvular interstitial cells. Using unbiased protein-RNA screening and molecular validation, we found that AVCAPIR directly interacts with FTO (fat mass and obesity-associated protein), subsequently blocking its N6-methyladenosine demethylase activity. Further transcriptomic and N6-methyladenosine modification epitranscriptomic screening followed by molecular validation confirmed that AVCAPIR hindered FTO-mediated demethylation of CD36 mRNA transcripts, thus enhancing CD36 mRNA stability through the N6-methyladenosine reader IGF2BP1 (insulin-like growth factor 2 mRNA binding protein 1). In turn, the AVCAPIR-dependent increase in CD36 stabilizes its binding partner PCSK9 (proprotein convertase subtilisin/kexin type 9), a procalcific gene, at the protein level, which accelerates the progression of AVC. We identified a novel piRNA that induced AVC through an RNA epigenetic mechanism and provide novel insights into piRNA-directed theranostics in CAVD.

Association of aortic valve size with the degree of aortic valve calcification in patients with severe aortic stenosis

Abstract Background The development and progression of aortic valve calcification (AVC) is a major contributor to the severity of aortic stenosis (AS). Currently, the 2021 ESC/EACTS Guidelines recommend the use of the Agatston AVC score to predict the likelihood of severe AS in patients with unclear echocardiographic AS severity. It has been shown that AS in women is associated with less AVC, thus lower AVC thresholds for the diagnosis of severe AS in women are recommended. It has furthermore been demonstrated that women generally have smaller hearts. However, whether small aortic valve (AV) size, independent from sex, is associated with less AVC in patients with severe AS has not been explored. Purpose The aim of this study was to assess the association of AV dimension and the degree of AVC in severe AS. Methods AVC was assessed on computed tomography using the Agatston score in consecutive patients with severe AS. AV diameter and indexed AV annulus area adjusted for body surface area were measured. To analyze the association with AVC, Spearman rank correlation as well as multivariate linear regression models adjusted for age, sex, left ventricular ejection fraction and cardiovascular risk factors were performed. Results In total 446 patients (mean age 81±6 years, 45% female) were included. Median Agatston Score was 2949 AU [2135-3986] in men and 1755 AU [1303-2571] in women. Mean AV diameter was 24±2mm and mean indexed annulus area was 248±43mm2. We observed a significant correlation between AV Agatston score and AV diameter (r=0.6; p<0.001 Figure 1 Panel A) as well as between AV Agatston score and indexed annulus area (r=0.5; p <0.001; Figure 1 Panel B). On multivariate analysis AV diameter (β=273; p<0.001), indexed annulus area (β=13; p<0.001) and sex (β=396; p<0.001) were independently associated with AVC by the Agatston score. In addition, separate regression analyses in men and women showed a significant association of AV diameter (β(men)=265; β(women)=275; both p<0.001) and indexed annulus area (β(men)=13; β(women)=11; both p<0.001) with the AV Agatston score. Hypertension, dyslipidemia, diabetes mellitus, smoking and chronic kidney disease showed no statistically significant association with the amount of AVC. Conclusion Our results indicate that patients with severe AS and larger AV dimensions present with more AVC on Agatston scoring, independent from sex. From surgical data, it is known that smaller aortic bioprostheses cause higher gradients, consequently we hypothesize here, that less calcification is required in small valves to cause significant narrowing. Thus, AVC cut-offs for the diagnosis of AS should be indexed for AV size rather than sex.

Introducing a novel ex-vivo calcification model for human aortic valves

Abstract Background Aortic stenosis (AS) is a narrowing of the aortic valve opening due to calcification and thickening of the leaflets. There are currently no effective pharmacological treatments for AS, and surgery or percutaneous interventions are the only therapeutic options. The cellular and molecular mechanisms underlying AS are complex and not fully understood due to the complexity and multi-factorial nature of AS and the absence of suitable test systems. In the current in-vitro models to study valvular calcification, isolated valvular interstitial cells (VICs) are cultured in 2D and in the absence of its native extracellular matrix. In addition, other cell types like valvular endothelial cells (VECs) and macrophages, which have been shown to impact the behavior of VICs, are absent. Purpose Our goal is to establish an ex-vivo calcification model for human aortic valves which maintains the original valve structure and cellular components. Methods Diseased aortic valves were harvested from patients undergoing aortic valve replacement. A 5mm x 7mm piece of this aortic valve tissue was dissected and embedded in agarose, then cut into 300 mm slices using a vibratome (Figure 1). In this way VICs remained in their valvular environment allowing the most native response of VICs to stimuli. The use of multiple slices from a single patient ensured proper internal controls. The slices were placed on a cell culture insert and cultured for 14 days. To induce calcification, the slices were cultured in the presence of 3 mM phosphate (n=23). As a control, valvular slices were cultured in the absence of 3mM phosphate (n=12). To validate if the calcification induced in the ex vivo culture system can be molecularly modulated the glucocorticoid dexamethasone was added to cultures containing 3mM phosphate (n=8). The slices were assessed for the presence of calcification using Alizarin Red staining and for the presence of osteogenic and inflammatory markers using (Immuno) histochemistry. Results In the presence of 3mM Phosphate, 74% of the slices demonstrated calcification, whereas no calcification was observed in all slices cultured without 3mM Phosphate (p<0.0001; Figure 2a). Furthermore, osteogenic genes such as Runx and alkaline phosphatase-1, as well as the inflammation marker NFkb were expressed in the calcified slices (Figure 2a). Dexamethason inhibited the calcification in the slices treated with 3 mM phosphate (Figure 2b). Conclusions We developed a novel ex-vivo calcification model for human aortic valves in which the initiation and progression of aortic valve calcification can be studied. In addition, the calcification can be modulated at a molecular level providing a unique opportunity to uncover the molecular signaling pathways involved in valvular calcification and to test compounds facilitating pre-clinical translational studies.Figure 1Figure 2

Abstract 12257: The Relationship Between Histological Features of Epicardial Adipose Tissue and Aortic Valve Calcification Assessed on Computed Tomography

Background: It has been hypothesized that epicardial adipose tissue (EAT) biologically contributes to the pathogenesis of cardiac diseases. However, it remains unclear how EAT correlates with the progression of aortic valve calcification (AVC). We evaluated if histological features of EAT are specifically related to AVC assessed on computed tomography (CT). Methods: We enrolled 35 patients undergoing cardiac CT examination before elective cardiac surgery (coronary artery bypass graft [CABG] and/or cardiac valve surgery), in which AVC was detected on CT. Each patient was evaluated with EAT volume, coronary calcium score (CCS), AVC score, and the presence of high-risk coronary plaque (HRP) on CT. During each cardiac surgery, histological samples for immunohistochemistry were obtained from EATs adjacent to the left anterior descending and right coronary arteries. Each patient was assessed with the numbers of CD-68 + and CD-11c + individual macrophages and osteocalcin + individual cells in a total of 6 random high-power fields (x400) of EAT samples. Results: In the histological findings of EAT, osteocalcin + cells showed a weak correlation with CD-68 + macrophages ( r = 0.40, p = 0.016) and a moderate correlation with CD-11c + macrophages ( r = 0.60, p = 0.0001). The AVC score on CT correlated with CD-11c + macrophages and osteocalcin + cells in EAT (Fig 1), whereas CCS had no correlation with such histological findings. On multivariate analysis adjusted for age, sex, CABG vs. non-CABG, EAT volume, CCS, and the presence of HRP, the AVC score independently correlated with increased CD-11c + macrophages (β = 0.58; p = 0.0036) and osteocalcin + cells (β = 0.59; p = 0.0021) in EAT. Fig 2 shows representative cases with different levels of AVC and EAT histology. Conclusions: The inflammatory and osteogenic activity in EAT significantly correlate with the progression of AVC, which may indicate a specific contribution of EAT to aortic valve degeneration through biological activities.

Aortic valve calcification is promoted by interleukin-8 and restricted through antagonizing CXCR2.

Inflammatory cytokines play a critical role in the progression of calcific aortic valve disease (CAVD), for which there is currently no pharmacological treatment. The aim of this study was to test the hypothesis that interleukin-8 (IL-8), known to be involved in arterial calcification, also promotes aortic valve calcification (AVC) and to evaluate whether pharmacologically blocking the IL-8 receptor, CXC motif chemokine receptor 2 (CXCR2), could be effective in preventing AVC progression. A cohort of 195 patients (median age 73, 74% men) diagnosed with aortic valve stenosis (severe in 16.9% of cases) were prospectively followed by CT for a median time of 2.6 years. A Cox proportional hazards regression analysis indicated that baseline IL-8 serum concentrations were associated with rapid progression of AVC, defined as an annualized change in the calcification score by CT ≥ 110 AU/year, after adjustment for age, gender, bicuspid anatomy and baseline disease severity. In vitro, exposure of primary human aortic valvular interstitial cells (hVICs) to 15 pg/ml IL-8 induced a two-fold increase in inorganic phosphate (Pi)-induced calcification. IL-8 promoted NFκB pathway activation, MMP-12 expression, and elastin degradation in hVICs exposed to Pi. These effects were prevented by SCH527123, an antagonist of CXCR2. The expression of CXCR2 was confirmed in hVICs and samples of aortic valves isolated from patients with CAVD, in which the receptor was mainly found in calcified areas, along with MMP-12 and a degraded form of elastin. Finally, in a rat model of chronic kidney disease-associated CAVD, SCH527123 treatment (1 mg/kg/day given orally for 11 weeks) limited the decrease in aortic cusp separation, the increase in maximal velocity of the transaortic jet, and the increase in aortic mean pressure gradient measured by echocardiography, effects that were associated with a reduction in hydroxyapatite deposition and MMP-12 expression in the aortic valves. Overall, these results highlight, for the first time, a significant role for IL-8 in the progression of CAVD by promoting calcification via a CXCR2-- and MMP-12-dependent mechanism that leads to elastin degradation, and identify CXCR2 as a promising therapeutic target for the treatment of CAVD.

Factors associated with the progression of aortic valve calcification in older adults

BackgroundAortic valve calcification (AVC) is a common valvular abnormality that predisposes to stenosis; AVC progression and factors associated with it remain unclear. We investigated the association of clinical factors and serum biomarkers with AVC progression in a population-based cohort of older adults. MethodsParticipants enrolled in both the Cardiovascular Abnormalities and Brain Lesion study (CABL; years 2005–2010) and the Subclinical Atrial Fibrillation And Risk of Ischemic Stroke study (SAFARIS;2014–2019) represent the study cohort. AVC was defined as bright dense echoes >1 mm in size on ≥1 cusps; each cusp was graded on a scale of 0 (normal) to 3 (severe calcification) at baseline and follow up. Serum biomarkers were measured at the time of follow-up assessment. Results373 participants (mean 68.1 ± 7.6 years of age, 146 M/ 227F) were included. 139 (37%) had AVC progression;93 (25%) had mild progression (1 grade), and 46 (12%) had moderate-severe progression (≥2 grades). The only significant clinical predictor of any progression was the use of anti-hypertensive medication which was associated with older age, higher BMI and more frequent hypertension, diabetes and hyperlipidemia. In multivariable analysis including biomarkers, transforming growth factor beta 1 (TGF-β1) was significantly associated with both all and moderate-severe AVC progression. ConclusionsA significant number of elderly subjects with AVC show progression of their valve disease; individual vascular risk factors are not associated with AVC progression, although a combined effect may exist. Higher levels of TGF-β1 are observed in individuals with AVC progression.

Lipoprotein(a): Its Association with Calcific Aortic Valve Stenosis, the Emerging RNA-Related Treatments and the Hope for a New Era in "Treating" Aortic Valve Calcification.

The treatment of patients with aortic valve calcification (AVC) and calcific aortic valve stenosis (CAVS) remains challenging as, until today, all non-invasive interventions have proven fruitless in preventing the disease's onset and progression. Despite the similarities in the pathogenesis of AVC and atherosclerosis, statins failed to show a favorable effect in preventing AVC progression. The recognition of lipoprotein(a) [Lp(a)] as a strong and potentially modifiable risk factor for the development and, perhaps, the progression of AVC and CAVS and the evolution of novel agents leading in a robust Lp(a) reduction, have rekindled hope for a promising future in the treatment of those patients. Lp(a) seems to promote AVC via a 'three hit' mechanism including lipid deposition, inflammation and autotaxin transportation. All of these lead to valve interstitial cells transition into osteoblast-like cells and, thus, to parenchymal calcification. Currently available lipid-lowering therapies have shown a neutral or mild effect on Lp(a), which was proven insufficient to contribute to clinical benefits. The short-term safety and the efficacy of the emerging agents in reducing Lp(a) have been proven; nevertheless, their effect on cardiovascular risk is currently under investigation in phase 3 clinical trials. A positive result of these trials will probably be the spark to test the hypothesis of the modification of AVC's natural history with the novel Lp(a)-lowering agents.

Lipoprotein(a) and progression of aortic valve calcification: a case of collider bias?

Lipoprotein(a) and progression of aortic valve calcification: a case of collider bias?

Lipoprotein(a) and progression of aortic valve calcification: a case of collider bias? Reply.

Lipoprotein(a) and progression of aortic valve calcification: a case of collider bias? Reply.

High-Density Lipoprotein and Long-Term Incidence and Progression of Aortic Valve Calcification: The Multi-Ethnic Study of Atherosclerosis.

Aortic valve calcification (AVC) shares pathological features with atherosclerosis. Lipoprotein components have been detected in aortic valve tissue, including HDL (high-density lipoprotein). HDL measures have inverse associations with cardiovascular disease, but relationships with long-term AVC progression are unclear. We investigated associations of HDL cholesterol, HDL-particle number and size, apoC3-defined HDL subtypes, and, secondarily, CETP (cholesteryl ester transfer protein) mass and activity, with long-term incidence and progression of AVC. We used linear mixed-effects models to evaluate the associations of baseline HDL indices with AVC. AVC was quantified by Agatston scoring of up to 3 serial computed tomography scans over a median of 8.9 (maximum 11.2) years of follow-up in the Multi-Ethnic Study of Atherosclerosis (n=6784). After adjustment, higher concentrations of HDL-C (high-density lipoprotein cholesterol), HDL-P (HDL particles), large HDL-P, and apoC3-lacking HDL-C were significantly associated with lower incidence/progression of AVC. Neither small or medium HDL-P nor apoC3-containing HDL-C was significantly associated with AVC incidence/progression. When included together, a significant association was observed only for HDL-C, but not for HDL-P. Secondary analyses showed an inverse relationship between CETP mass, but not activity, and AVC incidence/progression. In exploratory assessments, inverse associations for HDL-C, HDL-P, large HDL-P, and apoC3-lacking HDL with AVC incidence/progression were more pronounced for older, male, and White participants. ApoC3-containing HDL-C only showed a positive association with AVC in these subgroups. In a multiethnic population, HDL-C, HDL-P, large HDL-P, and apoC3-lacking HDL-C were inversely associated with long-term incidence and progression of AVC. Further investigation of HDL composition and mechanisms could be useful in understanding pathways that slow AVC.

Valve Endothelial Cell Exposure to High Levels of Flow Oscillations Exacerbates Valve Interstitial Cell Calcification.

The aortic valve facilitates unidirectional blood flow to the systemic circulation between the left cardiac ventricle and the aorta. The valve’s biomechanical function relies on thin leaflets to adequately open and close over the cardiac cycle. A monolayer of valve endothelial cells (VECs) resides on the outer surface of the aortic valve leaflet. Deeper within the leaflet are sublayers of valve interstitial cells (VICs). Valve tissue remodeling involves paracrine signaling between VECs and VICs. Aortic valve calcification can result from abnormal paracrine communication between these two cell types. VECs are known to respond to hemodynamic stimuli, and, specifically, flow abnormalities can induce VEC dysfunction. This dysfunction can subsequently change the phenotype of VICs, leading to aortic valve calcification. However, the relation between VEC-exposed flow oscillations under pulsatile flow to the progression of aortic valve calcification by VICs remains unknown. In this study, we quantified the level of flow oscillations that VECs were exposed to under dynamic culture and then immersed VICs in VEC-conditioned media. We found that VIC-induced calcification was augmented under maximum flow oscillations, wherein the flow was fully forward for half the cardiac cycle period and fully reversed for the other half. We were able to computationally correlate this finding to specific regions of the aortic valve that experience relatively high flow oscillations and that have been shown to be associated with severe calcified deposits. These findings establish a basis for future investigations on engineering calcified human valve tissues and its potential for therapeutic discovery of aortic valve calcification.

Dihydromyricetin ameliorates osteogenic differentiation of human aortic valve interstitial cells by targeting c-KIT/interleukin-6 signaling pathway.

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.

Lipoprotein(a) is associated with the onset but not the progression of aortic valve calcification.

Lipoprotein(a) [Lp(a)] is a potential causal factor in the pathogenesis of aortic valve disease. However, the relationship of Lp(a) with new onset and progression of aortic valve calcium (AVC) has not been studied. The purpose of the study was to assess whether high serum levels of Lp(a) are associated with AVC incidence and progression. A total of 922 individuals from the population-based Rotterdam Study (mean age 66.0±4.2 years, 47.7% men), whose Lp(a) measurements were available, underwent non-enhanced cardiac computed tomography imaging at baseline and after a median follow-up of 14.0 [interquartile range (IQR) 13.9-14.2] years. New-onset AVC was defined as an AVC score >0 on the follow-up scan in the absence of AVC on the first scan. Progression was defined as the absolute difference in AVC score between the baseline and follow-up scan. Logistic and linear regression analyses were performed to evaluate the relationship of Lp(a) with baseline, new onset, and progression of AVC. All analyses were corrected for age, sex, body mass index, smoking, hypertension, dyslipidaemia, and creatinine. AVC progression was analysed conditional on baseline AVC score expressed as restricted cubic splines. Of the 702 individuals without AVC at baseline, 415 (59.1%) developed new-onset AVC on the follow-up scan. In those with baseline AVC, median annual progression was 13.5 (IQR = 5.2-37.8) Agatston units (AU). Lipoprotein(a) concentration was independently associated with baseline AVC [odds ratio (OR) 1.43 for each 50 mg/dL higher Lp(a); 95% confidence interval (CI) 1.15-1.79] and new-onset AVC (OR 1.30 for each 50 mg/dL higher Lp(a); 95% CI 1.02-1.65), but not with AVC progression (β: -71 AU for each 50 mg/dL higher Lp(a); 95% CI -117; 35). Only baseline AVC score was significantly associated with AVC progression (P < 0.001). In the population-based Rotterdam Study, Lp(a) is robustly associated with baseline and new-onset AVC but not with AVC progression, suggesting that Lp(a)-lowering interventions may be most effective in pre-calcific stages of aortic valve disease.

Vitamin K2 and D in Patients With Aortic Valve Calcification: A Randomized Double-Blinded Clinical Trial.

Background:Menaquinone-7 (MK-7), also known as vitamin K2, is a cofactor for the carboxylation of proteins involved in the inhibition of arterial calcification and has been suggested to reduce the progression rate of aortic valve calcification (AVC) in patients with aortic stenosis.Methods:In a randomized, double-blind, multicenter trial, men from the community with an AVC score >300 arbitrary units (AU) on cardiac noncontrast computer tomography were randomized to daily treatment with tablet 720 µg MK-7 plus 25 µg vitamin D or matching placebo for 24 months. The primary outcome was the change in AVC score. Selected secondary outcomes included change in aortic valve area and peak aortic jet velocity on echocardiography, heart valve surgery, change in aortic and coronary artery calcification, and change in dp-ucMGP (dephosphorylated-undercarboxylated matrix Gla-protein). Safety outcomes included all-cause death and cardiovascular events.Results:From February 1, 2018, to March 21, 2019, 365 men were randomized. Mean age was 71.0 (±4.4) years. The mean (95% CI) increase in AVC score was 275 AU (95% CI, 225–326 AU) and 292 AU (95% CI, 246–338 AU) in the intervention and placebo groups, respectively. The mean difference on AVC progression was 17 AU (95% CI, –86 to 53 AU; P=0.64). The mean change in aortic valve area was 0.02 cm2 (95% CI, –0.09 to 0.12 cm2; P=0.78) and in peak aortic jet velocity was 0.04 m/s (95% CI, –0.11 to 0.02 m/s; P=0.21). The progression in aortic and coronary artery calcification score was not significantly different between patients treated with MK-7 plus vitamin D and patients receiving placebo. There was no difference in the rate of heart valve surgery (1 versus 2 patients; P=0.99), all-cause death (1 versus 4 patients; P=0.37), or cardiovascular events (10 versus 10 patients; P=0.99). Compared with patients in the placebo arm, a significant reduction in dp-ucMGP was observed with MK-7 plus vitamin D (–212 pmol/L versus 45 pmol/L; P<0.001).Conclusions:In elderly men with an AVC score >300 AU, 2 years MK-7 plus vitamin D supplementation did not influence AVC progression.Registration:URL: https://www.clinicaltrials.gov; Unique identifier: NCT03243890.

Iron promotes Slc7a11-deficient valvular interstitial cell osteogenic differentiation: A possible mechanism by which ferroptosis participates in intraleaflet hemorrhage-induced calcification

Calcific aortic valve disease (CAVD) is the most frequent pathogeny of aortic valve replacement in developed countries. Iron deposits are found in the intraleaflet hemorrhage (IH) areas of calcific aortic valves. Ferroptosis is a form of regulated cell death that involves metabolic dysfunction resulting from iron overload-dependent excessive lipid peroxidation. In this study, histological analysis showed that ferroptosis occurs in the IH areas of calcific aortic valves. We also demonstrated that Slc7a11 is expressed at low levels in OM-treated valvular interstitial cells (VICs) and IH areas and that low Slc7a11 expression is associated with calcification in CAVD. However, iron overload treatment did not promote VIC calcification under osteogenic conditions in vitro. Using lentiviral transfection to knockdown Slc7a11 in VICs, we found that the degree of iron overload-induced ferroptosis was positively increased in vitro. Finally, we also found that Slc7a11 knockdown promoted the osteogenic differentiation of VICs in vitro. In summary, this study reports a novel mechanism linking ferroptosis and CAVD development in which iron may promote Slc7a11-deficient VIC osteogenic differentiation by aggravating ferroptosis in vitro, thereby accelerating the progression of aortic valve calcification.

Bioinformatics and Machine Learning Methods to Identify FN1 as a Novel Biomarker of Aortic Valve Calcification

AimThe purpose of this study was to identify potential diagnostic markers for aortic valve calcification (AVC) and to investigate the function of immune cell infiltration in this disease.MethodsThe AVC data sets were obtained from the Gene Expression Omnibus. The identification of differentially expressed genes (DEGs) and the performance of functional correlation analysis were carried out using the R software. To explore hub genes related to AVC, a protein–protein interaction network was created. Diagnostic markers for AVC were then screened and verified using the least absolute shrinkage and selection operator, logistic regression, support vector machine-recursive feature elimination algorithms, and hub genes. The infiltration of immune cells into AVC tissues was evaluated using CIBERSORT, and the correlation between diagnostic markers and infiltrating immune cells was analyzed. Finally, the Connectivity Map database was used to forecast the candidate small molecule drugs that might be used as prospective medications to treat AVC.ResultsA total of 337 DEGs were screened. The DEGs that were discovered were mostly related with atherosclerosis and arteriosclerotic cardiovascular disease, according to the analyses. Gene sets involved in the chemokine signaling pathway and cytokine–cytokine receptor interaction were differently active in AVC compared with control. As the diagnostic marker for AVC, fibronectin 1 (FN1) (area the curve = 0.958) was discovered. Immune cell infiltration analysis revealed that the AVC process may be mediated by naïve B cells, memory B cells, plasma cells, activated natural killer cells, monocytes, and macrophages M0. Additionally, FN1 expression was associated with memory B cells, M0 macrophages, activated mast cells, resting mast cells, monocytes, and activated natural killer cells. AVC may be reversed with the use of yohimbic acid, the most promising small molecule discovered so far.ConclusionFN1 can be used as a diagnostic marker for AVC. It has been shown that immune cell infiltration is important in the onset and progression of AVC, which may benefit in the improvement of AVC diagnosis and treatment.

Sex Differences in Factors Associated With Progression of Aortic Valve Calcification in the General Population.

Guidelines recommend measurement of the aortic valve calcification (AVC) score to help differentiate between severe and nonsevere aortic stenosis, but a paucity exists in data about AVC in the general population. The aim of this study was to describe the natural history of AVC progression in the general population and to identify potential sex differences in factors associated with this progression rate. Noncontrast cardiac computed tomography was performed in 1298 randomly selected women and men aged 65 to 74 years who participated in the DANCAVAS trial (Danish Cardiovascular Screening). Participants were invited to attend a reexamination after 4 years. The AVC score was measured at the computed tomography, and AVC progression (ΔAVC) was defined as the difference between AVC scores at baseline and follow-up. Multivariable regression analyses were performed to identify factors associated with ΔAVC. Among the 1298 invited citizens, 823 accepted to participate in the follow-up examination. The mean age at follow-up was 73 years. Men had significantly higher AVC scores at baseline (median AVC score 13 Agatston Units [AU; interquartile range, 0-94 AU] versus 1 AU [interquartile range, 0-22 AU], P<0.001) and a higher ΔAVC (median 26 AU [interquartile range, 0-101 AU] versus 4 AU [interquartile range, 0-37 AU], P<0.001) than women. In the fully adjusted model, the most important factor associated with ΔAVC was the baseline AVC score. However, hypertension was associated with ΔAVC in women (incidence rate ratios, 1.58 [95% CI, 1.06-2.34], P=0.024) but not in men, whereas dyslipidemia was associated with ΔAVC in men (incidence rate ratio: 1.66 [95% CI, 1.18-2.34], P=0.004) but not in women. The magnitude of the AVC score was the most important marker of AVC progression. However, sex differences were significant; hence, dyslipidemia was associated with AVC progression only among men; hypertension with AVC progression only among women. Registration: URL: https://www.isrctn.com; Unique identifier: ISRCTN12157806.

Letter by Spiering et al Regarding Article, “Effect of Denosumab or Alendronic Acid on the Progression of Aortic Stenosis: A Double-Blind Randomized Controlled Trial”

HomeCirculationVol. 144, No. 22Letter by Spiering et al Regarding Article, “Effect of Denosumab or Alendronic Acid on the Progression of Aortic Stenosis: A Double-Blind Randomized Controlled Trial” Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyRedditDiggEmail Jump toFree AccessLetterPDF/EPUBLetter by Spiering et al Regarding Article, “Effect of Denosumab or Alendronic Acid on the Progression of Aortic Stenosis: A Double-Blind Randomized Controlled Trial” Wilko Spiering, MD, PhD, Willem P. Mali, MD, PhD and Pim A. de Jong, MD, PhD Wilko SpieringWilko Spiering https://orcid.org/0000-0002-2493-6407 Departments of Vascular Medicine (W.S.), University Medical Center Utrecht, The Netherlands. Search for more papers by this author , Willem P. MaliWillem P. Mali Radiology (W.P.M., P.A.d.J.), University Medical Center Utrecht, The Netherlands. Search for more papers by this author and Pim A. de JongPim A. de Jong Radiology (W.P.M., P.A.d.J.), University Medical Center Utrecht, The Netherlands. Search for more papers by this author Originally published29 Nov 2021https://doi.org/10.1161/CIRCULATIONAHA.121.055622Circulation. 2021;144:e334To the Editor:We read with interest the article by Pawade et al1 on the effects of denosumab or alendronate on the progression of aortic stenosis. The authors conclude from this randomized, controlled trial that both drugs did not affect progression of aortic valve calcification in patients with calcific aortic stenosis. They call for a search to find alternative pathways and mechanisms to identify disease-modifying therapies for the growing population of patients with this potentially fatal condition. We think that this trial does not justify abandoning bisphosphonate therapy as a potential treatment, but that it would have been better if the authors had chosen etidronate as the comparator instead of alendronate.Mineralization in soft tissues such as arteries and valves is controlled by endogenous inhibitors of hydroxyapatite formation, of which inorganic pyrophosphate is the most important. Bisphosphonates are nonhydrolyzable analogs of inorganic pyrophosphate, and cumulating evidence shows that they can inhibit vascular calcification. Nonnitrogenous bisphosphonates, like etidronate, are potent inhibitors of ectopic calcification, whereas nitrogenous bisphosphonates, like alendronate, have a high affinity for bone and are more potent inhibitors of osteoclasts. In an ex vivo model of calcification in intact aortic valves, it was shown that etidronate completely prevented aortic valve leaflet calcification at already very low concentrations.2Pseudoxanthoma elasticum is a heritable ectopic mineralization disorder with increased medial arterial calcification. In an animal model of pseudoxanthoma elasticum, etidronate, but not alendronate, significantly reduced calcification, indicating that the inhibiting effect on ectopic calcification of bisphosphonates is not just a class effect.3 In a double-blind, placebo-controlled, randomized, controlled trial in patients with pseudoxanthoma elasticum, we showed that etidronate reduced arterial calcification.4 However, in a randomized, controlled trial in patients with chronic kidney disease, alendronate did not decrease the progression of vascular calcification.5We think that the absence of any effect of alendronate on the progression of aortic stenosis seen in the study of Pawade et al is in line with earlier findings that alendronate with its low affinity for soft tissues is not the ideal bisphosphonate for inhibiting ectopic calcification.We would like to know why, with cumulating evidence that etidronate is very efficacious in inhibiting soft tissue calcification, the authors decided to choose alendronate and not etidronate in their clinical trial.Article InformationDisclosuresNone.FootnotesCirculation is available at www.ahajournals.org/journal/circ