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Objective: Wingless/integrase 5a（Wnt5a） pathway is known to regulate the osteogenesis. In this study, we aimed to clarify the role of Wnt5a pathway in aortic valve ectopic calcification. Methods: Human aortic valve interstitial cells（HAVICs） were obtained from calcified aortic valves of patients with calcific aortic valve stenosis（CAVS）. HAVICs were separated to CD34-negative and -positive cells by flow cytometry. We measured real-time PCR, alkaline phosphatase（ALP） activity, Alizarin Red S staining as an index of calcification. Immunohistochemical staining was performed to confirm the distribution of Wnt5a on calcified and normal aortic valves. Results: HAVICs, especially, CD34-negative cells are highly sensitive to tumor necrosis factor-α（ TNF-α, 30 ng/mL）, and which accelerated the Wnt5a gene expression. Wnt5a antagonist, box5, did not down-regulate HAVIC calcification induced by TNF-α. Further, Wnt5a agonist, foxy5, did not accelerated HAVIC calcification induced by TNF-α. There was no significant difference in the TNF-α-induced acceleration of ALP activity between the cells treated with foxy5 and box5. Furthermore, the proportion of Wnt5a positive cells was no difference between calcified and normal valves. Conclusions: We confirmed that Wnt5a pathway does not regulate the TNF-α-induced aortic valve calcification in HAVICs obtained from CAVS patients.

Objective Calcified aortic valvular disease is known as an inflammation-related process related to force. The purpose of this study was to determine whether micromechanical force could induce valve calcification of porcine valvular interstitial cells and to examine the role of integrin αvβ3 in valvular calcification by using a novel method: magnetic twisting cytometry. Methods Porcine valvular interstitial cells were cultured in vitro, and micromechanical force was applied to porcine valvular interstitial cells using magnetic twisting cytometry. Changes in calcification-related factors osteopontin and RUNX2 were detected. By using the calcification medium, the optimal magnetic twisting cytometry parameters for inducing valvular interstitial cell calcification were determined, and a magnetic twisting cytometry calcification promotion model was established. The role of αvβ3 in calcification was studied by using αvβ3 antagonists to block the function of αvβ3. Results Reverse transcription polymerase chain reaction assays showed that the expression of osteopontin was enhanced 30 minutes after 25G-1Hz 5 minutes of stimulation. Western blotting assays showed that the expression of osteopontin and RUNX2 was upregulated 24 hours after 25G-1Hz 5 minutes of stimulation. The optimal magnetic twisting cytometry parameter for inducing porcine valvular interstitial cell calcification was 25G-2Hz for 10 minutes. The expression of osteopontin and RUNX2 decreased significantly after the addition of αvβ3 antagonist. Clinically, patients with bicuspid aortic valves had high expression of RUNX2 and β3 in the aortic valve, and β3 significantly correlated with RUNX2. Conclusions By using magnetic twisting cytometry, we established a porcine valvular interstitial cell calcification model by micromechanical force stimulation and obtained the optimal parameters. Integrin αvβ3 plays a key role in the aortic valve calcification process.

Objective:We aimed to investigate the effect of ambulatory blood pressure (BP) on aortic valve calcification (AVC) and coronary artery calcification (CAC), which are subclinical atherosclerotic diseases., Methods:In this population-based, cross-sectional study, we assessed office BP, mean ambulatory BP (24-h, awake, and asleep), and variability of ambulatory BP, as determined by the coefficient of variation (awake and asleep). AVC and CAC were quantified using an Agatston score (>0) based on computed tomography scanning. We calculated relative risks (RRs) and 95% confidence intervals (CIs) with a 1-standard deviation increment in each BP index for the presence of AVC and CAC using a multivariate-adjusted Poisson regression with robust error variance., Results:Of 483 participants (mean age: 66.8 years), 154 (31.9%) and 310 (64.2%) had AVC and CAC, respectively. The presence of AVC was associated with office systolic BP (SBP; RR, 1.15; 95% CI, 1.03-1.28), awake diastolic BP (DBP) variability (RR, 1.12; 95% CI, 1.01-1.25), and asleep SBP variability (RR, 1.14; 95% CI, 1.03-1.27). The presence of CAC was associated with office SBP (RR, 1.08; 95% CI, 1.01-1.15), mean 24-h SBP (RR, 1.10; 95% CI, 1.04-1.16), mean awake SBP (RR, 1.11; 95% CI, 1.04-1.17), mean asleep SBP (RR, 1.07; 95% CI, 1.01-1.13), and asleep SBP variability (RR, 1.07; 95% CI, 1.01-1.13)., Conclusion:These findings highlight the association of ambulatory BP indices with both AVC and CAC, but with different effects on their presences.

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 cm 2 (95% CI, –0.09 to 0.12 cm 2 ; 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 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.

BackgroundCalcific 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 research, we attempted to clarify the role of ferroptosis in CAVD.MethodsThe level of ferroptosis in tissue and valvular interstitial cells (VICs) was assessed by the contents of 4-HNE, NADPH, ROS, and GSH, lipid peroxidation and mitochondrial morphology. The levels of calcification, iron accumulation and Slc7a11 expression in surgical aortic valve specimens were detected by Alizarin red or Von Kossa, Perl’s blue and immunohistochemical staining. The osteogenic differentiation of VICs was assessed by PCR and western blot analyses. Furthermore, RNA sequencing was used to detect potential differentially expressed genes between normal and osteogenic medium-treated (OM-treated) VICs.ResultsOur experiments demonstrated that ferroptosis occurred in the IH areas of calcific aortic valves. We also found that Slc7a11 was expressed at low levels in OM-treated VICs and IH areas. Finally, we demonstrated that iron promoted Slc7a11-deficient VICs osteogenic differentiation by aggravating ferroptosis in vitro.ConclusionIn conclusion, iron promotes Slc7a11-deficient VIC osteogenic differentiation by aggravating ferroptosis in vitro, thereby accelerating the progression of aortic valve calcification.StatementStatements and opinions expressed in the articles and communications herein are those of the author(s) and not necessarily those of the Editor(s), Society, or publisher, and the Editor(s), Society, and publisher disclaim any responsibility or liability for such material.Brief SummaryIn this work, two novel notions have been proposed. First, we reported that ferroptosis participated in the progression of CAVD. Second, this is the first cytology experiment of valvular interstitial cells (VICs) to clarify the mechanism by which intraleaflet hemorrhage aggravates calve calcification. This research provides new ideas and targets for alleviating the progression of CAVD, especially in patients who have calcified aortic valves without severe stenosis.

Aims This review aims to provide an update of available methods for imaging calcification activity and potential therapeutic options. Data Synthesis: Aortic valve calcification represents the most common heart valve condition requiring treatment among adults in Western societies. No medical therapies are proven to be effective in treating symptoms or reducing disease progression. Therefore, surgical or transcatheter aortic valve replacement remains the only available treatment option. Elevated circulating concentrations of lipoprotein(a) is strongly associated with degenerative aortic stenosis. This relationship was first observed in prospective observational studies, and the causal relationship was confirmed in genetic studies. Conclusions: new therapeutic targets have been identified and new imaging techniques could be used to test the effectiveness of new agents and further clarify the pathophysiology of AVS. No therapy that specifically lowers Lp (a) levels has been approved for clinical use.

Calcific aortic valve disease (CAVD) is an important health burden due to its increasing prevalence and lack of available approaches. Osteogenic transdifferentiation of aortic valve interstitial cells (AVICs) contributes to valve calcification. SRY-related HMG-box transcription factor 5 (SOX5) is essential for cartilage development. Whether SOX5 is involved in AVIC calcification has not been determined. This study aimed to explore the role of SOX5 in warfarin-induced AVIC calcification. Immunostaining showed decreased SOX5 in human calcific AV and warfarin induced mouse calcific AV tissues compared with human noncalcific AV and control mouse AV tissues. In calcific human AVICs (hAVICs) and porcine AVICS (pAVICs), both knockdown and overexpression of SOX5 inhibited calcium deposition and osteogenic marker gene expression. Protein expression assays and ChIP assays showed that overexpression of SOX5 led to increased recruitment of SOX5 to the SOX9 promoter and resulted in increased mRNA and protein expression of SOX9. Coimmunoprecipitation and immunofluorescence showed that SOX5 binds to SOX9 with its HMG domain in nucleus. Blue Native PAGE showed overexpression of SOX5 led to multimeric complex formation of SOX5 and resulted in decreased binding of SOX5 to SOX9 similar to the results of knockdown of SOX5. Further ChIP and western blotting assays showed that both knockdown and overexpression of SOX5 resulted in SOX9 initiating transcription of anti-calcific gene LRP6 in warfarin-treated pAVICs. Knockdown of LRP6 rescues the anti-calcification effect of SOX5 overexpression. We found that both loss and gain of function of SOX5 lead to the same phenotype: decreased warfarin induced calcification. The stoichiometry of SOX5 is crucial for cooperation with SOX9, SOX9 nuclear localization and subsequent binding of SOX9 to LRP6 promoter. These results suggest that SOX5 is a potential target for the development of anti-calcification therapy.

Low-dose computed tomography (LDCT) is predominantly applied in lung cancer screening programs. Tobacco smoking is the main risk factor for developing lung cancer but is also common for cardiovascular diseases, including aortic stenosis (AS). Consequently, an increased prevalence of cardiovascular diseases is expected in lung cancer screenees. Therefore, initial aortic valve calcification evaluation should be additionally performed on LDCT. The aim of this study was to estimate a calcium score (CS) cutoff point for clinically significant AS diagnosis based on LDCT, confirmed by echocardiographic examination. The study included 6631 heavy smokers who participated in a lung cancer screening program (MOLTEST BIS). LDCTs were performed on all individuals and were additionally assessed for aortic valve calcification with the use of CS according to the Agatston method. Patients with CS ≥ 900 were referred for echocardiography to confirm the diagnosis of AS and to evaluate its severity. Of 6631 individuals, 54 met the inclusion criteria and underwent echocardiography for confirmation and assessment of AS. Based on that data, receiver operating characteristic (ROC) curves of CS were plotted, and cutoff points for clinically significant AS diagnosis were established: A CS of 1758 for at least moderate AS had 85.71% (CI 65.36–95.02%) sensitivity and 75.76% (CI 58.98–87.17%) specificity; a CS of 2665 for severe AS had 87.5% (CI 73.89–94.54%) sensitivity and 76.92% (CI 49.74–91.82%) specificity. This is the first study to assess possible CS cutoff points for diagnosing clinically significant AS detected by LDCT in lung cancer screening participants. LDCT with CS assessment could enable early detection of patients with clinically significant AS and therefore identify patients who require appropriate treatment.

Impairment of extracellular and intracellular calcium homeostasis can have a damaging effect on the functioning of both individual organs and systems and the whole organism. In the cardiovascular system, disorders ofcalcium metabolism trigger a cascade of auto-inflammatory responses, leading to persistent morphological and functional disturbances. In this review, impaired calcium homeostasis is defined as a predictor of the development of hypersensitivity in cardiovascular diseases. We analyzed the development of a cardiovascular pathology (atherosclerosis, aortic valve calcification, and essential hypertension) with impaired calcium metabolism and pathological mechanisms that cause the disturbances. Calcium sensors, polymorphic variants of genes, and hormones involved in calcium metabolism can reveal the features of calcium metabolism and contribute to a personalized approach to treatment of cardiovascular diseases.

Aims Calcific aortic valve disease (CAVD) is frequent in the elderly. Telocytes (TCs) are implicated in intercellular communication by releasing extracellular vesicles (EVs). This study investigated the role of TC‐EVs in aortic valve calcification. Methods and results TCs were obtained and identified using enzymolysis method and flow cytometry. EVs were isolated from TCs using differential high‐speed centrifugation method and identified using transmission electron microscope, western blot, and qNano analysis. The mouse model of CAVD was established. The changes of aortic valve activity‐related indicators were analysed by ultrasound, and the expressions of TC markers CD34 and vimentin in mouse valve tissues were detected using RT‐qPCR and western blot. The model mice were injected with TC‐derived EVs. The expressions of Runx2, osteocalcin, and caspase‐3 were detected using RT‐qPCR and western blot. The calcification model of valvular interstitial cells (VICs) was established. TC‐EVs were co‐cultured with calcified VICs, and calcium deposition was detected using alizarin red S staining. miR‐30b expression in calcified valvular tissues and cells was detected after EV treatment. miR‐30b expression in TCs was knocked down and then EVs were extracted and co‐cultured with calcified VICs. The target of miR‐30b was predicted through bioinformatics website and verified using dual‐luciferase assay. The levels of Wnt/β‐catenin pathway‐related proteins were detected. ApoE−/− mice fed with a high‐fat diet showed decreased aortic valve orifice area, increased aortic transvalvular pressure difference and velocity, reduced left ventricular ejection fraction, decreased CD34 and vimentin, and increased caspase‐3, Runx2, and osteocalcin. The levels of apoptosis‐ and osteogenesis‐ related proteins were inhibited after EV treatment. TC‐EVs reduced calcium deposition and osteogenic proteins in calcified VICs. EVs could be absorbed by VICs. miR‐30b expression was promoted in calcified valvular tissues and cells after EV treatment. Knockdown of miR‐30b weakened the inhibitory effects of TC‐EVs on calcium deposition and osteogenic proteins. miR‐30b targeted Runx2. EV treatment inhibited the Wnt/β‐catenin pathway, and knockdown of miR‐30b in TCs attenuated the inhibitory effect of TC‐EVs on the Wnt/β‐catenin pathway. Conclusion TC‐EVs played a protective role in aortic valve calcification via the miR‐30b/Runx2/Wnt/β‐catenin axis.

Aims Aortic valve calcification (AVC) detected by non-contrast computed tomography (NCCT) associates with morbidity and mortality in patients with aortic valve stenosis. However, the importance of AVC in the general population is sparsely evaluated. We intend to describe the associations between AVC score on NCCT and echocardiographic findings as left atrial (LA) dilatation, left ventricular (LV) hypertrophy, aortic valve area (AVA), peak velocity, mean gradient, and aortic valve replacement (AVR) in a population with AVC scores ≥300 AU. Methods and results Of 10 471 males aged 65–74 years from the Danish Cardiovascular Screening trial (DANCAVAS), participants with AVC score ≥300 AU were invited for transthoracic echocardiography and 828 (77%) of 1075 accepted the invitation. AVC scores were categorized (300–599, 600–799, 800–1199, and ≥1200 AU). AVR was obtained from registries. AVC was significantly associated with a steady increase in LA dilation (10.5%, 16.3%, 15.8%, 19.6%, P = 0.031), LV hypertrophy (3.9%, 6.6%, 8.9%, 10.1%, P = 0.021), peak velocity (1.7, 1.9, 2.1, 2.8 m/s, P = 0001), mean gradient (6, 8, 11, 19 mmHg, P = 0.0001), and a decrease in AVA (2.0, 1.9, 1.7, 1.3 cm2, P = 0.0001). The area under the curve was 0.79, 0.93, and 0.92 for AVA ≤1.5 cm2, peak velocity ≥3.0 m/s, and mean gradient ≥20 mmHg, respectively, and the associated optimal AVC score thresholds were 734, 1081, and 1019 AU. AVC > 1200 AU was associated with AVR (P Conclusion Among males from the background population, increasing AVC scores were associated with LA dilatation, LV hypertrophy, AVA, peak aortic velocity, mean aortic gradient, and AVR.

Calcific aortic valve disease is a common heart disease that contributes to increased cardiovascular morbidity and mortality. There is a lack of effective pharmaceutical therapy because its mechanisms are not yet fully known. Ginkgo biloba extract (EGB761) is reported to alleviate vascular calcification. However, whether EGB761 protects against aortic valve calcification, a disease whose pathogenesis shares many similarities with vascular calcification, and potential molecular mechanisms remain unknown. In this study, porcine aortic valve interstitial cell (pAVIC) calcification was induced by warfarin with or without the presence of EGB761. Immunostaining was performed to establish and characterize the pAVIC phenotype. Calcium deposition and calcium content were examined by Alizarin Red S staining and an intracellular calcium content assay. Alkaline phosphatase activity was detected by the p-nitrophenyl phosphate method. The expression levels of bone morphogenetic protein-2 (BMP2), Runt-related transcription factor 2 (Runx2), homeobox protein MSX-2, and phosphorylated (p)-Smad1/5 were detected by reverse transcription-quantitative polymerase chain reaction (PCR) and Western blot analysis. Consistent with these in vitro data, we also confirmed the suppression of in vivo calcification by EGB761 in the warfarin-induced C57/Bl6 mice. The results indicated that both pAVICs and aortic valves tissue of mice stimulated with warfarin showed increased calcium deposition and expression of osteogenic markers (alkaline phosphatase, BMP2, homeobox protein MSX-2, and Runx2) and promoted p-Smad1/5 translocation from the cytoplasm to the nucleus. The addition of EGB761 significantly inhibited p-Smad1/5 translocation from the cytoplasm to the nucleus, thus suppressing calcification. In conclusion, EGB761 could ameliorate warfarin-induced aortic valve calcification through the inhibition of the BMP2-medicated Smad1/5/Runx2 signaling pathway.

Background and Aims: Mutations of the NT5E gene encoding the cluster of differentiation 73 (CD73) protein have been found in patients with characteristic calcification of joints and arteries (CALJA). CD73 plays a protective role against aortic valve calcification and its deletion results in aortic valve calcification. However, there have been no reports of a patient with CALJA with aortic stenosis. Methods: We describe two extremely rare cases of two sisters with identical NT5E gene mutation patterns, both of whom presented severe aortic stenosis and limb ischemia. Genetic examination for definitive diagnosis was also performed in both patients. Results: Both patients underwent aortic valve replacement and bilateral distal arterial bypass surgeries successfully. They were genetically diagnosed with CALJA based on the NT5E mutation. Conclusions: NT5E mutation should be considered in patients requiring aortic valve replacement for a calcified aortic valve and bypass surgery for specific calcified and occluded arteries.

ObjectiveAortic valve calcification (AVC) and coronary artery calcification (CAC) are predictors of cardiovascular disease (CVD), presumably sharing risk factors. Our objectives were to determine the prevalence and extent of AVC in a large population of men aged 60–74 years and to assess the association between AVC and cardiovascular risk factors including CAC and biomarkers.MethodsParticipants from the DANish CArdioVAscular Screening and intervention trial (DANCAVAS) with AVC and CAC scores and without previous valve replacement were included in the study. Calcification scores were calculated on non-contrast CT scans. Cardiovascular risk factors were self-reported, measured or both, and further explored using descriptive and regression analysis for AVC association.Results14 073 men aged 60–74 years were included. The AVC scores ranged from 0 to 9067 AU, with a median AVC of 6 AU (IQR 0–82). In 8156 individuals (58.0%), the AVC score was >0 and 215 (1.5%) had an AVC score ≥1200. In the regression analysis, all cardiovascular risk factors were associated with AVC; however, after inclusion of CAC ≥400, only age (ratio of expected counts (REC) 1.07 (95% CI 1.06 to 1.09)), hypertension (REC 1.24 (95% CI 1.09 to 1.41)), obesity (REC 1.34 (95% CI 1.20 to 1.50)), known CVD (REC 1.16 (95% CI 1.03 to 1.31)) and serum phosphate (REC 2.25 (95% CI 1.66 to 3.10) remained significantly associated, while smoking, diabetes, hyperlipidaemia, estimated glomerular filtration rate and serum calcium were not.ConclusionsAVC was prevalent in the general population of men aged 60–74 years and was significantly associated with all modifiable cardiovascular risk factors, but only selectively after adjustment for CAC ≥400 AU.Trial registration numberNCT03946410 and ISRCTN12157806.

Calcific aortic valve disease (CAVD) is a common progressive disease of the aortic valves, for which no medical treatment exists and surgery represents currently the only therapeutic solution. The development of novel pharmacological treatments for CAVD has been hampered by the lack of suitable test-systems, which require the preservation of the complex valve structure in a mechanically and biochemical controllable system. Therefore, we aimed at establishing a model which allows the study of calcification in intact mouse aortic valves by using the Miniature Tissue Culture System (MTCS), an ex vivo flow model for whole mouse hearts. Aortic valves of wild-type mice were cultured in the MTCS and exposed to osteogenic medium (OSM, containing ascorbic acid, beta-glycerophosphate and dexamethasone) or inorganic phosphates (PI). Osteogenic calcification occurred in the aortic valve leaflets that were cultured ex vivo in the presence of PI, but not of OSM. In vitro cultured mouse and human valvular interstitial cells calcified in both OSM and PI conditions, revealing in vitro-ex vivo differences. Furthermore, endochondral differentiation occurred in the aortic root of ex vivo cultured mouse hearts near the hinge of the aortic valve in both PI and OSM conditions. Dexamethasone was found to induce endochondral differentiation in the aortic root, but to inhibit calcification and the expression of osteogenic markers in the aortic leaflet, partly explaining the absence of calcification in the aortic valve cultured with OSM. The osteogenic calcifications in the aortic leaflet and the endochondral differentiation in the aortic root resemble calcifications found in human CAVD. In conclusion, we have established an ex vivo calcification model for intact wild-type murine aortic valves in which the initiation and progression of aortic valve calcification can be studied. The in vitro-ex vivo differences found in our studies underline the importance of ex vivo models to facilitate pre-clinical translational studies.

The objective of our study was to determine the relationship between exposure to environmental tobacco smoke (ETS) and the value of the aortic valve calcification score (AVCS) in people suffering from arterial hypertension (AH). 107 non-smokers with AH (mean age 67.16 ± 8.48 years) were qualified for the study. The degree of exposure to ETS was assessed using the Second-hand Smoke Exposure Scale (SHSES) questionnaire. Study group was divided depending on ETS exposure: A—no exposure, B—low, C—medium and D—high. AVCS was measured based on the aortic valve plane multiplanar reconstruction from the non-contrast phase of the cardiac computed tomography. The Agatston algorithm was used, in which calcifications were considered changes with a density exceeding 130 HU. The mean AVCS value in the study group of patients was 213.59 ± 304.86. The AVCS was significantly lower in subgroup A than in subgroups C and D. In subgroup A, the lack of aortic valve calcification (AVCS = 0) was observed significantly more frequently than in subgroups C and D. There was a positive correlation between the number of SHSES points and the AVCS value (r = 0.37, p

Background:Risk factors for atherosclerotic disease including dyslipidemia have been shown to be associated with aortic valve calcification (AVC). Nuclear magnetic resonance (NMR)-measured lipoprotein particles, low-density and high-density lipoprotein particles (LDL-p, HDL-p) in particular, have emerged as novel markers of atherosclerotic disease; however, whether NMR-measured particles are associated with AVC remains to be determined. This study aimed to examine the association between NMR-based lipoprotein particle measurements and standard lipids with AVC. The primary variables of interest were LDL-p (nmol/L), HDL-p (μmol/L), LDL-cholesterol, and HDL-cholesterol (both in mg/dL).Methods and Results:A community-based random sample of Japanese men aged 40-79 years examined in 2006-2008, in Shiga, Japan was studied. Presence of AVC was defined as an Agatston score >0. Lipoprotein particles were measured using NMR spectroscopy. In the main analysis, multivariable-adjusted odds ratios (ORs) and 95% confidence intervals (95% CIs) for the prevalence of AVC across the higher quartiles of lipids in reference to the lowest ones were obtained. Of 874 participants analyzed, 153 men had AVC. Multivariable-adjusted ORs of prevalent AVC for the highest vs. the lowest quartile were significantly elevated for LDL-p (OR, 2.20; 95% CI: 1.23-3.93) and LDL-cholesterol (OR, 2.16; 95% CI: 1.23-3.78). In contrast, neither HDL-p nor HDL-cholesterol was associated with AVC., Conclusions:The association of prevalent AVC with NMR-based LDL-p was comparable to that with LDL-cholesterol.

Objectives Optimizing valve implantation depth (ID) plays a crucial role in minimizing conduction disturbances and achieving optimal functional integrity. Until now, the impact of intraprocedural fast (FP) or rapid ventricular pacing (RP) on the implantation depth has not been investigated. Therefore, we aimed to (1) evaluate the impact of different pacing maneuvers on ID, and (2) identify the independent predictors of deep ID. Methods 473 TAVR patients with newer-generation self-expanding devices were retrospectively enrolled and one-to-one propensity-score-matching was performed, resulting in a matching of 189 FP and RP patients in each cohort. The final ID was analyzed, and the underlying functional, anatomical, and procedural conditions were evaluated by univariate and multivariate analysis. Results The highest ID was reached under RP in severe aortic valve calcification and valve size 26 mm. Multivariate analysis identified left ventricular outflow (LVOT) calcification [OR 0.50 (0.31–0.81) p = 0.005*], a “flare” aortic root [OR 0.42 (0.25–0.71), p = 0.001*], and RP (OR 0.49 [0.30–0.79], p = 0.004*) as independent highly preventable predictors of a deep ID. In a model of protective factors, ID was significantly reduced with the number of protective criteria (0–2 criteria: − 5.7 mm ± 2.6 vs. 3–4 criteria − 4.3 mm ± 2.0; p Conclusion Data from this retrospective analysis indicate that RP is an independent predictor to reach a higher implantation depth using self-expanding devices. Randomized studies should prove for validation compared to fast and non-pacing maneuvers during valve delivery and their impact on implantation depth. Trail registration Clinical Trial registration: NCT01805739. Graphic abstract Study design: Evaluation of the impact of different pacing maneuvers (fast ventricular pacing—FP vs. rapid ventricular pacing—RP) on implantation depth (ID). After one-to-one-propensity-score-matching, independent protective and risk factors for a very deep ID beneath 6 mm toward the LVOT (®. AVC aortic valve calcification.

Background Our goal was to determine the accuracy of 3-dimensional transesophageal echocardiography (3D-TEE) compared with that of computed tomography (CT) in the preoperative evaluation for transcatheter aortic valve replacement (TAVR) when the errors caused by inconsistent software and method have been eliminated and the representativeness of the sample has been improved. We also investigated the influence of aortic root calcification on the accuracy of 3D-TEE in aortic annulus evaluations. Methods Part I: 45 of 233 patients who underwent TAVR in the department of cardiovascular surgery at the Xijing hospital from January 2016 to August 2019 were studied retrospectively. Materialise Mimics software and the multiplanar reconstruction method were used for evaluation, based on 3D-TEE and CT. The annulus area-derived diameter, the annulus perimeter-derived diameter (Dp), the annulus mean diameter, the left ventricular outflow tract Dp diameter, the sinotubular junction (STJ) diameter-Dp, and the aortic sinus diameter were compared and analyzed. Part II: 31 of 233 patients whose 3D-TEE and CT data were well preserved and in the required format were included. HU450 and HU850 were used as indicators to measure the severity of calcification. The Spearman rank correlation and Linear regression were used to analyze the correlation between aortic root calcification and the accuracy of 3D-TEE in aortic annulus measurement. Results The measurement results based on 3D-TEE were significantly lower than those obtained using CT (P P = 0.11). The correlation coefficient of the two groups was 0.699–0.954 (P Conclusions Although an evaluation based on 3D-TEE underestimated the results, we can deduce CT results from 3D-TEE because the two methods exhibit considerable correlation and consistency. Trial registration Name: Surgery and Transcatheter Intervention for Structural Heart Diseases. Number: NCT02917980. URL: https://clinicaltrials.gov/ct2/results?term=NCT02917980.

Little is known about the biological functions and underlying mechanisms of long non-coding RNA AFAP1-AS1 in degenerative calcified aortic valve disease (DCAVD). This study aims to explore whether AFAP1-AS1 regulates macrophage polarization in aortic valve calcification. Macrophage polarization and AFAP1-AS1 expression were detected in normal and calcified aortic valves of DCAVD patients. To explore the effect of AFAP1-AS1 on macrophage polarization, gain and loss of function were performed in THP-1 cells, and the percentage of M1 and M2 and the expressions of M1 and M2 markers were analyzed. Meanwhile, osteogenic differentiation was examined in valve interstitial cells (VICs). Compared with normal valves, there were more M1, less M2, and high AFAP1-AS1 expressions in calcified aortic valves, which may indicate a relationship between AFAP1-AS1 and macrophage polarization. AFAP1-AS1 overexpression promoted M1 polarization in lipopolysaccharide (LPS) and interferon gamma (IFN-γ)-treated THP-1 cells but inhibited M2 polarization, as well as augmented VIC osteogenic differentiation. On the contrary, the silence of AFAP1-AS1 could induce macrophage to M2-type and inhibit VIC osteogenic differentiation. These results elucidate that AFAP1-AS1 can promote M1 macrophages polarization to aggravate VIC osteogenic differentiation, playing a role in aortic valve calcification.

We investigated aortic valve calcification (AVC) distribution and predictors for leaflet calcification patterns in patients with severe tricuspid aortic valve stenosis undergoing transcatheter aortic valve replacement (TAVR). Patients undergoing routine multi-sliced computed tomography (MSCT) for procedural planning were enrolled. MSCT data were transferred to a dedicated workstation for evaluation (3mensio Structural Heart™, Pie Medical Imaging BV, Maastricht, The Netherlands) and analyzed. Participants were separated into asymmetrical (AC) and symmetrical (SC) leaflet calcification and potential predictors for calcification distribution were identified with univariate and multivariate regression analysis. 567 Participants with severe tricuspid AS were divided into asymmetrical (AC, n = 443; 78.1%) and symmetrical (SC, n = 124; 21.9%) AVC. In AC, the non-coronary cusp was the most calcified cusp (n = 238; 57.7%). SC is more common in females (AC/SC: 49.2% vs. 67.7%; p 67° (OR 1.68 [1.00–2.80], p = 0.049) as predictors for SC, although with only moderate predictive value. Data from this retrospective analysis indicate that SC occurs more frequently in females. The cumulative leaflet calcification burden is higher in patients with AC, who also present with larger aortic root dimensions. The predictive value for prominent calcification of different aortic valve cusps in AC patients was only low to moderate. Trial registration number: NCT01805739. Supplementary Information The online version contains supplementary material available at 10.1007/s10554-021-02248-6.

Background. Aortic valve stenosis is common with prevalence of about 0.5 %, peaking in people aged over 70 years mostly due to age-related valve calcification. The year 2002 was marked by the invention and use of the endovascular aortic replacement valve by an A. Cribier’s group of French surgeons. Russian endovascular surgery introduced transcatheter aortic valve replacement in 2009, having since built an extensive experience in this practice. Perioperative mortality in patients under 70 years with no serious comorbidity ranges from 1 to 3 %, however, reaching two-fold 4–8 % in elderly patients. The emergence of minimally invasive technologies offered cure to critical patients, who would merely not get over an open surgery.Materials and methods. This case study provides video recordings of transcatheter aortic valve replacement (Accurate Neo) in transfemoral approach performed for the first time in the Republic of Bashkortostan. Patient K., 70 yo, diagnosis: Atherosclerosis. Aortic valve stenosis. FC III. Complications: aortic valve calcification st. III, CHF II A, FC III, persistent atrial fibrillation, tachysystole. Comorbid: CHD. Exertional angina. FC III. CHF II A, FC III.Results and discussion. Improving the transcatheter valve type facilitates an optimal individual aortic valve selection. Pre-replacement valvuloplasty was performed in all patients. The valve replacement is followed by transoesophageal echocardiography to justify possible aortic valve post-dilatation upon marked paravalvular regurgitation. The implant positioning relative to the aortic valve fibrous crown and mitral valve flaps is precisely controlled with ultrasound.Conclusion. Interventional radiology currently provides high-quality, effective, minimally invasive medical aid even in aortic stenosis patients with multiple comorbidity. In the patient’s denial of open surgery, transcatheter aortic valve replacement represents a sole alternative treatment, also increasing the life expectancy and quality. A wider diversity of available transcatheter devices enables a better personalisation of the biological valve replacement procedure.

Background: While an association between atherosclerosis and dementia has been identified, few studies have assessed the longitudinal relationship between aortic valve calcification (AVC) and cognitive impairment (CI). Objective: We sought to determine whether AVC derived from lung cancer screening CT (LCSCT) was associated with CI in a moderate-to-high atherosclerotic risk cohort. Methods: This was a single site, retrospective analysis of 1401 U.S. veterans (65 years [IQI: 61, 68] years; 97%male) who underwent quantification of AVC from LCSCT indicated for smoking history. The primary outcome was new diagnosis of CI identified by objective testing (Mini-Mental Status Exam or Montreal Cognitive Assessment) or by ICD coding. Time-to-event analysis was carried out using AVC as a continuous variable. Results: Over 5 years, 110 patients (8%) were diagnosed with CI. AVC was associated with new diagnosis of CI using 3 Models for adjustment: 1) age (HR: 1.104; CI: 1.023–1.191; p = 0.011); 2) Model 1 plus hypertension, hyperlipidemia, diabetes, CKD stage 3 or higher (glomerular filtration rate

Background Aortic valve stenosis （AVS） is the most common heart valve disease in elderly society. The effective treatment for AVS is surgical aortic valve replacement but with extremely invasive. Although transcatheter aortic valvereplacement also is performed for highly risk patients with minimally invasive, it has to resolve the problem ofbioprosthetic valve durability in future. However, there is no effective medical treatment established for AVS toinhibit acceleration of aortic valve calcification. Objectives We aimed to confirm whether the ascending aorta is the best location for AVS to innovate local and selective medicaltherapy or not. Methods and Results After anesthetized by isoflurane, the wild type rat was stablished on operating table. Opening chest to heart bymedian sternotomy approach, the catheter was inserted into left ventricular from apical for body perfusion. Weinjected saturated solution of amido black 10B （AB） into ascending aorta above sino-tubular junction about 0.5 cmfrom aortic valves without aortic dissection observed, and body perfusion was performed by saline containing heparinin order to prevent embolism happened in vascular. After 30 minutes, the heart with the ascending aorta wasextracted. We used optical microscope to check the existence of AB at around of aortic valves or not. Interestingly,AB was detected at around of aortic valves and valsalva sinus of the aorta and myocardium in all rats. Conclusions These results suggested that AB injected is delivered from the ascending aorta to aortic valves through vasavasorum. These data provide very important evidences for local medical therapy development for AVS patients.

Cardamonin (CDM) is a natural chalcone with strong anti-inflammatory properties. Inflammation-induced osteogenic changes in valve interstitial cells (VICs) play crucial roles in the development of calcific aortic valve disease (CAVD), a degenerative disease characterized by degeneration, thickening, fibrosis, and calcification of the heart valve tissues. To investigate the anti-osteogenic differentiation role of CDM in human valve interstitial cells (hVICs), which consequently reverses the calcification of the aortic valve, human VICs were exposed to osteogenic induction medium (OM) with CDM for further cell viability, osteogenic gene and protein expression analyses, and anti-calcification testing. mRNA sequencing was utilized to analyze the differentially expressed genes (DEGs) and related signaling pathways as potential molecular targets involved in CDM's anti-calcification activity. Human aortic valve leaflet ex vivo calcific cultures were used to investigate the CDM inhibition of osteogenic differentiation of hVICs at the tissue level. ApoE-/- mice fed with a high-fat (HF) diet were used to evaluate the effect of CDM on aortic valve calcification. No significant CDM cytotoxicity was seen in the hVICs at 10 μM. The addition of CDM to OM prevented calcified nodule accumulation, and a decrease in the gene/protein expression levels of BMP2, RUNX2, SPP1, TNF-α, and COL1A2 was observed. Venn diagram analysis of the DEGs identified 666 common DEGs and highlighted the NOD-like receptor signaling pathway (ko04621) as an anti-calcification target of CDM. CDM also repressed the activation of p-AKT, p-ERK1/2, and p-IκBα, and prevented the OM-induced nuclear transcription of NF-κB p65. In the in vitro and ex vivo calcific conditional culture experiments, CDM exhibited anti-inflammatory and anti-calcification effects by suppressing the activation of the NLRP3 inflammasome and downregulating IL-1β expression. In vivo, CDM ameliorated aortic valve calcification by interfering with NLRP3 expression. Our study demonstrated that CDM inhibited the phenotypical calcific transformation of hVICs by mediating the inactivation of the NF-κB/NLRP3 inflammasome. Therefore, it is considered to be a promising natural compound for use in preventing the progression of heart valve calcification disease.

Visual Abstract, Highlights • ECM proteins play an important role in maintaining the structural architecture and the mechanical behavior of the aortic valve. • Network analysis highlights a strong connection between metabolic markers and ECM proteins. • MXRA5 and FNDC1 were identified as new biomarkers of aortic stenosis in 2 independent cohorts, Summary This study analyzed the expression of extracellular matrix (ECM) proteins during aortic valve calcification with mass spectrometry, and further validated in an independent human cohort using RNAseq data. The study reveals that valve calcification is associated with significant disruption in ECM and metabolic pathways, and highlights a strong connection between metabolic markers and ECM remodeling. It also identifies FNDC1 and MXRA5 as novel ECM biomarkers in calcified valves, electing them as potential targets in the development and progression of aortic stenosis.

Background The CoreValve Evolut R and PRO (Medtronic, Minneapolis, MN, USA) are among the newest-generation of self-expandable transcatheter aortic valve replacement (TAVR) devices and show excellent results. Treating patients with severely calcified (SC) native aortic valve anatomy may be challenging because of the increased risk of periprocedural complications. This study investigated the performance of Evolut R and PRO in this special patient subset. Methods Patients who underwent TAVR with the CoreValve Evolut R or PRO (n=381) from September 2015 to March 2018 were divided by aortic valve calcification extent. Patients with SC aortic valve anatomy (n=98; men, >2,062 and women, >1,377 Agatston units) were compared with those with non-severely calcified (NCS) aortic valve anatomy after 1:2 propensity score matching. Outcomes were evaluated according to the updated valve academic research consortium criteria. Results Patients with SC anatomy were older (83 years vs 80 years, p Conclusion The CoreValve Evolut R and PRO showed good clinical safety profiles and excellent haemodynamic results in patients with SC anatomy and who more often required permanent pacemaker implantation.

Calcific aortic valve disease (CAVD) is a common heart valve disease in aging populations, and aberrant osteogenic differentiation of valvular interstitial cells (VICs) plays a critical role in the pathogenesis of ectopic ossification of the aortic valve. miR-214 has been validated to be involved in the osteogenesis process. Here, we aim to investigate the role and mechanism of miR-214 in CAVD progression. miR-214 expression was significantly downregulated in CAVD aortic valve leaflets, accompanied by upregulation of osteogenic markers. Overexpression of miR-214 suppressed osteogenic differentiation of VICs, while silencing the expression of miR-214 promoted this function. miR-214 directly targeted ATF4 and Sp7 to modulate osteoblastic differentiation of VICs, which was proved by dual luciferase reporter assay and rescue experiment. miR-214 knockout rats exhibited higher mean transvalvular velocity and gradient. The expression of osteogenic markers in aortic valve leaflets of miR-214 knockout rats was upregulated compared to that of the wild-type group. Taken together, our study showed that miR-214 inhibited aortic valve calcification via regulating osteogenic differentiation of VICs by directly targeting ATF4 and Sp7, indicating that miR-214 may act as a profound candidate of targeting therapy for CAVD., Graphical Abstract, miR-214 imbalance in aortic valve leaflets is responsible for the progress of calcific aortic valve disease (CAVD). Downregulation of miR-214 eliminates its inhibitory effect on translation of Sp7 and ATF4, thereby promoting the development of CAVD. These results suggested that miR-214 might provide an avenue for treating CAVD.

Background There is conflicting data regarding the association between low levels of plasma vitamin D and ischemic heart disease. We aimed to investigate the relationship between plasma vitamin D levels and heart valve calcification in hospitalized patients with ischemic heart disease versus non-ischemic heart disease controls. Methods A prospective case-control study comprising two age and gender-matched groups. The study group included consecutive patients hospitalized due to acute coronary syndrome; the control group included consecutive non-ischemic heart disease patients hospitalized for noncardiac causes. Blood samples for 25-hydroxyvitamin D level were drawn. An echocardiogram was performed during the first 3 days of hospitalization and reviewed for presence and degree of valvular calcification. Results Forty patients with acute coronary syndrome and 40 controls (age 58 ± 11 years, 64% male in both groups) were included. Mean plasma 25-hydroxyvitamin D vitamin level in the entire cohort was 24.5 ± 8 ng/ml. Valve calcification rates were similar in acute coronary syndrome versus non-acute coronary syndrome group (28 vs. 21 had valvular calcification; 18 vs. 12 had aortic valve calcification; 21 vs. 14 had mitral valve calcification, respectively; P = NS for all). We found no significant relationship between vitamin D level and valvular calcification, aortic valve calcification, or mitral valve calcification rate or degree in the entire cohort and in each group alone (P = NS for all). There was a negative correlation between 25-hydroxyvitamin D levels and age in the acute coronary syndrome group (r = -0.399, P = 0.012). Conclusions We did not find a significant relationship between plasma vitamin D levels and the rate or degree of calcification of either aortic/mitral/both valves in hospitalized patients with or without ischemic heart disease.

Aortic valve calcification (AVC) determined by computed tomography has emerged as a complementary measure of aortic stenosis (AS) severity and as a predictor of adverse events. Thus, AVC can guide further treatment decisions in patients with low-gradient AS (LG-AS). We compared the symptomatic and prognostic outcome of patients with low vs. high AVC after transcatheter aortic valve implantation (TAVI). Patients with an aortic valve area index ≤ 0.6 cm2/m2 and a mean pressure gradient (MPG) 35 ml/m2, LVEF ≥ 50%, n = 244); patients with MPG ≥ 40 mmHg (n = 1142) served as controls. Patients were further categorized according to published AVC thresholds. Demographic characteristics and cardiovascular risk were not different between patients with high vs. low AVC in any of the subgroups. Patients with low AVC had a lower MPG. Symptom improvement at 30 days was observed in the majority of patients but was less pronounced in LFLG-AS patients with low vs. those with high AVC. Kaplan-Meier 1-year survival curves were identical between patients with low and high AVC in all three LG-AS groups. The severity of LG-AS based on AVC has no impact on 1-year prognosis once TAVI has been performed. • Aortic valve calcification (AVC) determined by computed tomography has emerged as a complementary measure of aortic stenosis (AS) severity and is of prognostic value in selected patients. • Patients with inconsistent echocardiographic measures can be classified as having severe or nonsevere AS by the computed tomography–derived AVC score. • The prognostic value of AVC in patients with low-gradient AS is abrogated after correction of afterload by TAVI.

Despite the focus placed on cardiovascular research, the prevalence of vascular and valvular calcification is increasing and remains a leading contributor of cardiovascular morbidity and mortality. Accumulating studies provide evidence that cardiovascular calcification is an inflammatory disease in which innate immune signaling becomes sustained and/or excessive, shaping a deleterious adaptive response. The triggering immune factors and subsequent inflammatory events surrounding cardiovascular calcification remain poorly understood, despite sustained significant research interest and support in the field. Most studies on cardiovascular calcification focus on innate cells, particularly macrophages' ability to release pro-osteogenic cytokines and calcification-prone extracellular vesicles and apoptotic bodies. Even though substantial evidence demonstrates that macrophages are key components in triggering cardiovascular calcification, the crosstalk between innate and adaptive immune cell components has not been adequately addressed. The only therapeutic options currently used are invasive procedures by surgery or transcatheter intervention. However, no approved drug has shown prophylactic or therapeutic effectiveness. Conventional diagnostic imaging is currently the best method for detecting, measuring, and assisting in the treatment of calcification. However, these common imaging modalities are unable to detect early subclinical stages of disease at the level of microcalcifications; therefore, the vast majority of patients are diagnosed when macrocalcifications are already established. In this review, we unravel the current knowledge of how innate and adaptive immunity regulate cardiovascular calcification; and put forward differences and similarities between vascular and valvular disease. Additionally, we highlight potential immunomodulatory drugs with the potential to target calcification and propose avenues in need of further translational inquiry.

BACKGROUND AND AIMS: Calcific aortic valve disease is highly prevalent in patients with significant smoking history and is a marker of atherosclerosis. The aim of this study was to define the prognostic value of aortic valve calcification (AVC) derived from low dose, lung cancer screening computed tomography (LCSCT) for all-cause mortality in this higher risk population. METHODS: This is a single site, retrospective analysis of 1529 moderate-to-high atherosclerotic cardiovascular risk U.S. veterans (65 years [IQI: 61, 68] years; 96% male), who underwent clinically indicated LCSCT. CTs were scored for aortic valve calcification (AVC) and coronary artery calcification (CAC). The primary endpoint was all-cause mortality and secondary endpoints were nonfatal myocardial infarction (MI) and nonfatal cerebrovascular accident (CVA). RESULTS: Over 4-year follow-up, 227 patients (15%) died, 112 patients (7%) had nonfatal MI, and 52 patients (3%) had nonfatal CVA. AVC was predictive of all-cause mortality (HR per 100: 1.041 [1.030–1.052], p < 0.001), and this association remained significant after multivariate adjustment for traditional atherosclerotic risk factors, including CAC (1.021 [1.007–1.036], p = 0.003). After excluding patients with severe aortic stenosis (AS) or severe AVC (≥1274 AU in women and ≥2065 AU in men), in a subset of 765 patients who had echocardiograms, this association remained significant after multivariate analysis (HR per 100: 1.052 [1.010–1.095], p = 0.014). Despite controlling for CAC in the models, AVC was still associated with MI (HR per 100: 1.021 [1.004–1.039], p = 0.017) and with CVA (HR per 100: 1.027 [1.002–1.051], p = 0.032). CONCLUSIONS: Scoring AVC derived from LCSCT is predictive of mortality, nonfatal MI, and nonfatal CVA in patients at known risk for cardiovascular disease, independent of coronary calcification or severe aortic valve stenosis.

Smoking is a major risk factor for both cardiovascular disease (CVD) and lung cancer. Aortic valve calcification (AVC) and coronary artery calcification (CAC) are both due to atherosclerotic disease. We aim to investigate whether AVC on low-dose CT (LDCT) predicts death from CVD in smokers beyond that provided by CAC. We reviewed a prospective cohort of 8618 smokers enrolled in LDCT screening for lung cancer in New York State between June 2000 and December 2005. As of December 2009, 169 of the 643 deaths were due to CVD; median follow-up time was 96.4 months. Visual AVC was assessed as being absent (AVC = 0) or present (AVC > 0). CAC ordinal scores of 0–12 were categorized into three validated prognostic categories (0, 1–3, and 4–12). Cox proportional hazards regression analysis was used to assess whether AVC > 0 increased the risk of CVD death, after adjustment for CAC categories and other risk factors. The prevalence of AVC significantly increased (p 0 and CAC ≥ 4, the hazard ratio was 2.35 (95%CI 1.57–3.50) compared with the reference group of AVC = 0 and CAC 0 and CAC ≥ 4 had more than a 2-fold increased risk of CVD death than the group with AVC = 0 and CAC

Graphical abstract, Highlights • H2S derived from CSE/CBS inhibits inflammation and calcification of valvular interstitial cells. • H2S releasing molecules inhibits inflammation and calcification of valvular interstitial cells. • H2S inhibits inflammation and calcification in aortic valve of apolipoprotein E deficient mice. • H2S suppresses nuclear translocation of NF-κB and subsequent expression of IL-1β and TNF-α. • Activation of Runx2, its nuclear translocation is mediated by NF-κB in calcifying conditions. • Inflammation and calcification are connected via master transcription factors, NF-κB and Runx2., Introduction Hydrogen sulfide (H2S) was revealed to inhibit aortic valve calcification and inflammation was implicated in the pathogenesis of calcific aortic valve disease (CAVD). Objectives We investigate whether H2S inhibits mineralization via abolishing inflammation. Methods and results Expression of pro-inflammatory cytokines, interleukin-1β (IL-1β) and tumor necrosis factor α (TNF-α) were increased in patients with CAVD and in calcified aortic valve of ApoE-/- mice. Administration of H22S releasing donor (4-methoxyphenyl piperidinylphosphinodithioc acid (AP72)) exhibited inhibition on both calcification and inflammation in aortic valve of apolipoprotein E knockout mice (ApoE-/-) mice is reflected by lowering IL-1β and TNF-α levels. Accordingly, AP72 prevented the accumulation of extracellular calcium deposition and decreased nuclear translocation of nuclear factor-κB (NF-κB) in human valvular interstitial cells (VIC). This was also accompanied by reduced cytokine response. Double-silencing of endogenous H2S producing enzymes, Cystathionine gamma-lyase (CSE) and Cystathionine beta-synthase (CBS) in VIC exerted enhanced mineralization and higher levels of IL-1β and TNF-α. Importantly, silencing NF-κB gene or its pharmacological inhibition prevented nuclear translocation of runt-related transcription factor 2 (Runx2) and subsequently the calcification of human VIC. Increased levels of NF-κB and Runx2 and their nuclear accumulation occurred in ApoE-/- mice with a high-fat diet. Administration of AP72 decreased the expression of NF-κB and prevented its nuclear translocation in VIC of ApoE-/- mice on a high-fat diet, and that was accompanied by a lowered pro-inflammatory cytokine level. Similarly, activation of Runx2 did not occur in VIC of ApoE-/- mice treated with H2S donor. Employing Stimulated Emission Depletion (STED) nanoscopy, a strong colocalization of NF-κB and Runx2 was detected during the progression of valvular calcification. Conclusions Hydrogen sulfide inhibits inflammation and calcification of aortic valve. Our study suggests that the regulation of Runx2 by hydrogen sulfide (CSE/CBS) occurs via NF-κB establishing a link between inflammation and mineralization in vascular calcification.

Background Compared to high gradient aortic stenosis (AS), patients with low-flow, low-gradient AS have higher mortality after transcatheter aortic valve replacement (TAVR), but distinct outcome predictors in this patient subset are yet to be determined. The present study investigated the prognostic impact of aortic valve calcification (AVC) in patients with low-flow, low-gradient AS undergoing TAVR. Methods This retrospective single-center analysis includes all patients undergoing TAVR for severe low-flow, low-gradient AS (n = 526), ie, low EF low gradient AS (LEF-LG AS; n = 290) and paradoxical low-flow, low-gradient AS (PLF-LG AS; n = 236), in whom AVC was quantified from contrast-enhanced multislice computed tomography images. AVCdensity was defined as calcium volume per annulus area. Patients were trichotomized according to sex-specific AVCdensity tertiles in both subgroups. All-cause mortality was assessed by Kaplan-Meier analyses and independent outcome predictors were determined by multivariable analyses. Results In both subgroups, patients with high AVCdensity had higher mean transvalvular gradients at baseline and higher rates of PVL after TAVR. High AVCdensity was associated with lowest 1- and 3-year mortality after TAVR in the LEF-LG AS but not in the PLF-LG AS group. According to multivariable analysis AVCdensity was independently associated with better survival in LEF-LG AS patients (HR 0.73 [0.60-0.88], P = .0011), but not in those with PLF-LG AS (HR 0.91 [0.73-1.14], P = .42). Conclusions Quantification of AVC may not only be of diagnostic but also of prognostic value, as it facilitates the selection of LEF-LG AS patients with higher probability of beneficial outcome after TAVR.

Background: Echocardiography-derived calcium score (ECS) was able to predict the presence of extensive coronary calcium and obstructive Coronary artery disease(CAD), assessed using Multi slice CT (MSCT) coronary angiography. Furthermore, significant linear trends were observed between ECS and extent and severity of coronary atherosclerosis and number of calcified coronary artery lesions. Objective: To correlate an echocardiography-derived calcium score (ECS), obtained using coronary artery calcium Score (CACS) and the presence of obstructive CAD. Patients and Methods: The study comprised 100 patients (males and females) presented with symptoms suspected of stable CAD. All cases were recruited from the outpatient clinic in Agouza police Hospital in Cairo and Islamic center of Al-Azhar University during a period of fourteen months from 1/11/2018 to 30/12/2019. Results: Linear regression analysis identified that the most independent variables that can predict AVC were CAC score, dyslipidemia, and the most independent variable that can predict obstructive coronary artery disease was the presence of coronary artery calcification and higher CAC score. ROC curve analysis was used to test the predictive value of aortic valve calcification (AVC), Mitral annulus calcification (MAC) and CAC scores for the presence or absence of obstructive CAD. This analysis showed that AVC and MAC scores were inferior to CAC score which was found to have the highest predictive value evidenced by an area under the curve of 0.763 (95%CI: 0.781, 0.913). MAC score was inferior to AVC score which was found to have the highest predictive value evidenced by an area under the curve of 0.815 (95%CI: 0.758, 0.879). Conclusion: The echocardiographic calcium scores specifically the MVC and AVC was correlated to be CAC. ECS can predict the CAC score. ECS can predict the presence or absence CAD.

Aims The aims of this study were to investigate the correlation and sex differences between total valve calcium, valve calcium concentration, and aortic valve calcification (AVC) in explanted valves from patients with severe aortic valve stenosis undergoing aortic valve replacement (AVR). Methods and results Sixty-nine patients with severe aortic stenosis (AS) scheduled for elective AVR underwent echocardiography and cardiac computed tomography (CT) prior to surgery (AVCin vivo) and CT of the explanted aortic valve (AVCex vivo). Explanted valves were prepared in acid solution, sonicated, and analysed with Arsenazo III dye to estimate total valve calcium and valve calcium concentration. Median AVCex vivo was 2082 (1421–2973) AU; mean valve calcium concentration was 1.43 ± 0.42 µmol Ca2+/mg tissue; median total valve calcium 156 (111–255) mg Ca2+, and valve calcium density 52 (35–81) mg/cm2. AVC displayed a strong correlation with total valve calcium (R2 = 0.98, P Conclusion AVC score provides a strong estimate for total valve calcium but to a lesser degree calcium concentration in the valve tissue of patients with severe AS. Females presented lower valvular calcium concentrations than males irrespective of AS severity, adding evidence and providing support to the important point that sex differences in valvular calcium concentration in AS does not reflect valvular size.

Background Growing evidence has indicated that microRNAs (miRNAs) are involved in the progression of calcific aortic valve disease (CAVD), a progressive pathological condition with no effective pharmacological therapy. This study was set out with the aim to investigate possible roles of miR-195 in CAVD. Methods and results Initially, the differential expressed genes (DEGs) associated with CAVD were screened out and miRNAs potentially regulating VWF were predicted from microarray analysis. Next, we quantified VWF and miR-195 expression in isolated aortic valve interstitial cells (AVICs) and aortic valve tissues, followed by confirmation of the target relationship between miR-195 and VWF using the dual luciferase reporter assay. Furthermore, we evaluated the biological functions of miR-195 and VWF on ALP activity, cell differentiation, and the levels of miR-195, VWF, Runx2, OCN, ALP, p38 and phosphorylated p38 in AVICs. VWF was highly expressed, while miR-195 was poorly expressed in CAVD. Furthermore, miR-195 targeted VWF and negatively regulated its expression. Upregulation of miR-195 or silencing VWF could reduce ALP activity, calcified deposition, and the mRNA and protein levels of Runx2, OCN, and ALP by inhibiting the p38-MAPK signaling pathway, thereby ameliorating aortic valve calcification in vitro. Conclusions On all accounts, miR-195 can potentially inhibit aortic valve calcification by repressing VWF and p38-MAPK signaling pathway, highlighting a theoretical basis for pharmacological treatment of CAVD.

Aortic valve calcification severity has been associated with higher rates of aortic regurgitation (AR) following TAVR, but scarce data exist on its impact with the use of newer generation transcatheter heart valves.This was a multicenter study including 626 patients with severe aortic stenosis who underwent TAVR with the SAPIEN 3 valve. Patients were divided in 2 groups according to the median index calcium score (iCS) for each sex: high CS (HCS, iCS ≥ median), and low iCS (LCS, iCS median). Another analysis was performed in those patients with extreme iCS (ECS, iCS75th percentile for each sex). Clinical and echocardiographic data were collected prospectively in a dedicated database.The mean CS was 3758 ± 1417 AU and 1616 ± 691 AU in the HCS and LCS groups, respectively (p 0.001). There were no differences between groups in 30-day mortality (HCS:2.6%, LCS:1.0%, p = 0.13) and stroke (HCS:2.6%,LCS:2.6%, p = 1.0) rates, but all cases (n = 5) of annulus rupture occurred in the HCS group (1.6% vs. 0%, p = 0.061). The incidence of moderate-severe AR post-TAVR was low in both groups (HCS:1.6%,LCS:1.6%, p = 1.0), and valve gradient and area were similar between groups. The results remained similar in the ECS group (mean CS:4607 ± 1424 AU), but a mildly increased mean transvalvular gradient post-TAVR was observed in ECS patients (12.1 ± 5.6 vs 11.0 ± 4.3 mmHg; p = 0.015).Aortic valve calcification severity failed to impact mortality/stroke rates following TAVR with the SAPIEN 3 valve. Low rates of significant AR were observed irrespective of CS, and a mild increase in transvalvular gradient was observed in ECS patients.

Purpose Cardiovascular mortality remains high among patients with diabetes compared with the general population. The primary aim was to evaluate the interest in and demand for advanced cardiovascular screening in patients with diabetes; the secondary aim was to explore its efficiency in detecting unprotected subclinical cardiovascular disease (CVD). Patients and methods In a cross-sectional design, randomly selected 40-60-year-old men and women with diabetes were invited to the screening trial. Screening encompassed (1) a comprehensive medical interview; (2) non-contrast computed tomography scanning to quantify coronary artery and aortic valve calcification, to measure left atrial size, to assess heart rhythm and to detect aortic and iliac dilatations; (3) ankle and brachial blood pressure measurements; and (4) blood and urine samples for measurements of HbA1c, lipid profile, renal function, NT-pro B-type natriuretic peptide (pro-BNP) and albuminuria. Primary outcome was participation rate; secondary outcome was rate of unprotected subclinical CVD. Results Of 465 invited patients, 191 (41.1%) attended screening. The participation rate was 40% (95% CI:33-47) for males and 42% (95% CI:36-48) for females. Twenty-four patients were excluded due to previous CVD. The remaining patients' mean age was 52 years; 58% were males. Subclinical CVD was found in 64%, with a male preponderance (males 75% (95% CI:66-83; females 49% (95% CI:37-60)). Presence of severe coronary artery calcification (score ≥ 400) showed a male preponderance (males 19% (95% CI:12-27); females 7% (95% CI:3-16)). Aortic valve calcification, enlarged left atrial volume, atrial fibrillation, aortic dilatations, peripheral artery disease or increased pro-BNP were uncommon, and without any sex differences. Unprotected subclinical CVD was very common, and medical treatment was intensified in 60% (95% CI:53-68) of patients. Conclusion We propose a feasible cardiovascular screening examination from which middle-aged patients with diabetes may benefit. However, the participation rate may be too low to warrant screening.

Aortic valve stenosis (AS) is the third most common cardiovascular disease. The prevalence of both AS and arterial hypertension increases with age, and the conditions therefore often co-exist. Co-existence of AS and arterial hypertension is associated with higher global left ventricular (LV) pressure overload, more abnormal LV geometry and function, and more adverse cardiovascular outcome. Arterial hypertension may also influence grading of AS, leading to underestimation of the true AS severity. Current guidelines suggest re-assessing patients once arterial hypertension is controlled. Management of arterial hypertension in AS has historically been associated with prudence and concerns, mainly related to potential adverse consequences of drug-induced peripheral vasodilatation combined with reduced stroke volume due to the fixed LV outflow obstruction. Current evidence suggests that patients should be treated with antihypertensive drugs blocking the renin–angiotensin–aldosterone system, adding further drug classes when required, to achieve similar target blood pressure (BP) values as in hypertensive patients without AS. The introduction of transcatheter aortic valve implantation has revolutionized the management of patients with AS, but requires proper BP management during and following valve replacement. The purpose of this document is to review the recent evidence and provide practical expert advice on management of hypertension in patients with AS.

Inflammation is implicated in the pathogenesis of calcific aortic valve disease (CAVD) which is a major contributor to cardiovascular mortality and lacks non-surgical treatment. The progranulin (PGRN) is an important immunomodulatory factor in a variety of inflammatory diseases, including rheumatoid arthritis, osteoarthritis, inflammatory bowel disease and pneumonia. However, its role in calcification of aortic valve remains unknown. We firstly found that PGRN was increased in calcified human aortic valve (AV) tissues. Interestingly, in addition to full-length PGRN (68KD), a much stronger band of approximately 45 KD was also significantly increased. The band of 45 KD (45-GRN), was present in wild type (WT) mouse MEFs and AV but absent in grn-/-MEFs, indicating that it was a specific degradation product derived from PGRN. 45-GRN was upregulated whereas PGRN was reduced in human valve interstitial cells (hVICs) under calcifying conditions which is induced by osteogenic medium (OM). In primary porcine VICs (pVICs), PGRN downregulated TNF-α and α-SMA which was accompanied by downregulation of RUNX2, OPN, OCN, alkaline phosphatase activity and calcium deposition, effects pointing to reduced inflammation, myofibroblastic and osteoblastic transition under calcifying conditions. We overexpressed a mimic of 45-GRN which contains p-G-F-B-A-C in pVICs. However, 45-GRN overexpression promoted OM-induced calcification through activating the Smad1/5/8, NF-κB and AKT signaling pathways. Inhibition of the three signaling pathways suppressed 45-GRN's effect on VICs phenotype transition. 45-GRN promoted TNF-α and expressed converse pathogenic signatures with PGRN during TNF-α stimulation. Collectively, this study provides new insight into the pathogenesis of CAVD, indicating that PGRN is a stratagem in mitigating valve fibrosis/osteoblastic differentiation, and also presenting 45-GRN as a potential target for the treatment of CAVD.

Reduced bioavailability of nitric oxide (NO) has been implicated in the pathogenesis of calcific aortic stenosis. Herein, we investigated the effects of l-Arginine, the main precursor of NO, on the osteogenic differentiation of aortic interstitial valve cells (VICs).We isolated a clonal population of bovine VICs that expresses osteogenic markers and induces calcification of collagen matrix after stimulation with endotoxin (LPS 500 ng/mL). VICs were treated in vitro with different combinations of LPS ± l-Arginine (50 or 100 mM) and cell extracts were collected to perform proteomic (iTRAQ) and gene expression (RT-PCR) analysis.l-Arginine prevents the over-expression of alkaline phosphatase (ALP, p 0.001) and reduces matrix calcification (p 0.05) in VICs treated with LPS. l-Arginine also reduces the over-expression of inflammatory molecules induced by LPS (TNF-alpha, IL-6 and IL-1beta, p 0.001). The proteomic analysis allowed to identify 49 proteins with an altered expression profile after stimulation with LPS and significantly modified by l-Arginine. These include proteins involved in the redox homeostasis of the cells (i.e. Xanthine Oxidase, Catalase, Aldehyde Oxidase), remodeling of the extracellular matrix (i.e. ADAMTSL4, Basigin, COL3A1) and cellular signaling (i.e. Fibrillin-1, Legumain, S100A13). The RT-PCR analysis confirmed the modifications of Fibrillin-1, ADAMTSL4, Basigin and Xanthine Oxidase, whose expression levels increase after stimulation with LPS and are reduced by l-Arginine (p 0.05).l-Arginine prevents osteogenic differentiation of VICs and reduces matrix calcification. This effect is achieved through the modulation of proteins involved in the cellular redox system, remodeling of extracellular matrix and inflammatory activation of VICs.

Aortic valve stenosis is the most prevalent heart valve disease worldwide. Although interventional treatment options have rapidly improved in recent years, symptomatic aortic valve stenosis is still associated with high morbidity and mortality. Calcific aortic valve stenosis is characterized by a progressive fibro-calcific remodeling and thickening of the aortic valve cusps, which subsequently leads to valve obstruction. The underlying pathophysiology is complex and involves endothelial dysfunction, immune cell infiltration, myofibroblastic and osteoblastic differentiation, and, subsequently, calcification. To date, no pharmacotherapy has been established to prevent aortic valve calcification. However, novel promising therapeutic targets have been recently identified. This review summarizes the current knowledge of pathomechanisms involved in aortic valve calcification and points out novel treatment strategies.

Aortic valve calcification leads to the deposition of calcium phosphate minerals in the extracellular matrix of the aortic valve leaflets. The mineral deposits can severely narrow the opening of the aortic valve, leading to aortic stenosis. There are no therapies to halt or slow down disease progression and the mechanisms governing aortic valve calcification are still poorly understood. Recently, several studies have shown that for the same aortic stenosis severity, women present significantly lower calcification loads than men. The cause of this sex-related difference is unknown. To understand this difference, we analyzed mineral deposits from surgically excised calcified human aortic valves with different material characterization techniques. We find profound differences in mineral composition and morphology between sexes, which strongly suggest that minerals form slower in women than in men and follow a different mineralization pathway. This finding paves the way for new approaches specifically geared towards men or women in the diagnosis and treatment of aortic valve calcification. STATEMENT OF SIGNIFICANCE: Aortic valve calcification is a health disorder with increasing prevalence and high morbidity and mortality. Currently there is no approved effective treatment; the only available therapeutic option is invasive valve replacement, to which not all patients are suited. The main reason for such lack of treatment options is our lack of understanding of the calcification mechanism. In this study, we show profound differences in mineral composition and morphology between sexes, suggesting that aortic valve calcification follows different mineralization pathways in men and women. These findings pave the way for new approaches specifically geared towards men or women in the diagnosis and treatment of aortic valve calcification.

To evaluate risk factors for prevalence and progression of aortic valve calcification (AVC) in lung cancer screening participants and also to assess the sensitivity and reliability of visual AVCs on low-dose CT (LDCT) for predicting aortic stenosis (AS) in high-risk smokers. We reviewed 1225 consecutive participants in annual LDCT screening for lung cancer at the Mount Sinai Hospital between 2010 and 2017. Sensitivity and specificity of moderate/severe AVC score on LDCT to identify AS on echocardiogram were calculated for 126 participants who had both within 12 months. Using regression analyses, risk factors for AVC at baseline, for progression, and for new AVC on annual rounds of screening were identified. Reliability of AVC assessment on LDCT was assessed by comparing visual AVC scores (1) with standard-dose, electrocardiography (ECG)-gated CT for 31 participants who had both within 12 months and (2) with Agatston scores of 1225 participants and by determining (3) the intra-reader agreement of 1225 participants. Visual AVC scores on LDCT had substantial agreement with the severity of AS on echocardiography and substantial inter-observer and excellent intra-observer agreement. Sensitivity and specificity of moderate/severe visual AVC scores for moderate/severe AS on echocardiogram were 100% and 94%, respectively. Significant predictors for baseline AVC were male sex (OR = 2.52), age (OR10 years = 2.87), and coronary artery calcification score (OR = 1.18), the significant predictor for AVC progression after baseline was pack-years of smoking (HR10 packyears = 1.14), and significant predictors for new AVC on annual LDCT were male sex (HR = 1.51), age (HR10 years = 2.17), CAC (HR = 1.09) and BMI (HR = 1.06). AVC scores on LDCT should be documented, especially in lung cancer screening program. • LDCT screening for lung cancer provides an opportunity to identify lung cancer and cardiovascular disease in asymptomatic smokers. • Visual aortic valve calcification scores could be reliably evaluated on LDCT and had substantial agreement with the severity of aortic valve stenosis on echocardiography. • Sensitivity and specificity of moderate/severe visual AVC scores on LDCT for moderate/severe AS on echocardiogram were 100% and 94%, respectively.

International audience; Aortic valve stenosis remains the most frequent structural heart disease, especially in the elderly. During the last decade, we noticed an important consideration and a huge number of publications related to the medical and surgical treatment of this disease. However, the molecular aspect of this degenerative issue has also been more widely studied recently. As evidenced in oncologic but also cardiac research fields, the emergence of microRNAs in the molecular screening and follow-up makes them potential biomarkers in the future, for the diagnosis, follow-up and treatment of aortic stenosis. Herein, we present a review on the implication of microRNAs in the aortic valve disease management. After listing and describing the main miRNAs of interest in the field, we provide an outline to develop miRNAs as innovative biomarkers and innovative therapeutic strategies, and describe a groundbreaking pre-clinical study using inhibitors of miR-34a in a pre-clinical model of aortic valve stenosis.

Prediction of patients at risk of aortic valve stenosis (AS), AS progression rate, and aortic bioprosthesis dysfunction are of major importance for clinical management and/or prevention. Many imaging modalities may be used; however, they may not be conclusive or available for all patients. Circulating biomarkers are easily available and may be related to a disease or process such as aortic valve calcification or associated with a risk factor of the disease. This article reviews current blood biomarkers associated with aortic valve stenosis/calcification and bioprosthesis dysfunction.

Severe aortic stenosis (AS) is associated with a progressive cardiac remodeling that ultimately leads to heart failure and death if the valve is not replaced. The confirmation of AS severity is therefore crucial to adequately manage patients with AS. Transthoracic echocardiography is the first-line examination to confirm AS severity, but it may be inconclusive in discordant cases. In this setting, AS severity should be confirmed using a multimodality imaging approach. This review gives an overview of how to assess and/or confirm AS severity, especially in case of discordance.