Search

Background Aortic valve calcification (AVC) has been known as an independent predictor for adverse cardiovascular events and all-cause mortality. Previous studies demonstrated that AVC was associated with aortic valve inflammation and atherosclerosis. However, the relationship between the progression of AVC and pericoronary inflammation remains undetermined. Purpose The purpose of this study was to evaluate the impact of the pericoronary inflammation on the progression of AVC. Methods A total of 107 patients with suspected or known chronic coronary syndromes who underwent clinically indicated serial 320-slice coronary computed tomography angiography (CTA) at Tsuchiura Kyodo General Hospital from January 2011 to June 2019 were retrospectively studied. Pericoronary inflammation was assessed by pericoronary adipose tissue attenuation (PCATA) defined as the mean CT attenuation value of PCATA (−190 to −30 Hounsfield units [HU]) on proximal 40 mm segments of coronary arteries. AVC was quantified by Agatston score on CTA. The mean aortic attenuation (HU Aorta) and the standard deviation (SD) in the region of interest at the level of the sinotubular junction was measured. AVC was defined as the threshold for calcium detection (mean HU Aorta + 2SD). AVC index was calculated as follows: (follow-up/baseline) AVC divided by follow-up period. AVC progression was defined as newly-developed AVC at follow-up or an increased AVC index during follow-up. All patients were divided into two groups according to the presence or absence of AVC progression, and clinical characteristics and CT findings were compared between these two groups. Results AVC progression was observed in 26 patients (24.3%) between 2 serial CT examinations (median, 34 months). There was no significant difference in age, gender and the prevalence of other cardiovascular risk factors between the 2 groups. Patients in AVC progression group were associated with higher prevalence of elevated PCATA-LAD, higher LV mass index at baseline and the initial AVC presence. Receiver-operating characteristic curve analysis revealed that the optimal cut off value of PCATA-LAD for predicting AVC progression was −68.26 HU (area under the curve 0.605; 95% confidence interval [CI], 0.465–0.745). Multivariable logistic regression analysis revealed that baseline PCATA-LAD ≥−68.26 HU (odds ratio [OR], 3.12; 95% CI, 1.04–9.35, p=0.042) and the presence of baseline positive AVC (OR, 6.84; 95% CI, 2.34–20.0, p=0.0004) were independent predictors of AVC progression. Conclusions The increased pericoronary inflammation and the presence of AVC may help identify patients with high risk for future AVC progression. Funding Acknowledgement Type of funding source: None

Background It is unclear whether the annual progression of aortic valve calcification (AVC) is associated with the progression of coronary atherosclerosis. Purpose We explored the association between AVC and the total and compositional plaque volume (PV) progression. Methods We performed a prospective multinational registry of consecutive patients who underwent serial coronary computed tomography angiography (CTA) at ≥2-year intervals. AVC, and total and compositional PV at baseline and follow-up were quantitatively analyzed. Multivariate linear regression models were constructed. Results Overall, 594 patients (56% male, 61.5±9.7 years old) were included (mean coronary CTA interval, 3.9±1.5 years). At baseline, AVC was 30.9±117.3. Normalized total PV at baseline was 122.3±219.4mm3, encompassing 41.9±116.8mm3 of calcified PV and 80.4±131.5mm3 of non-calcified PV. After adjustment of age, sex, clinical risk factors, and drug use, AVC at baseline was independently associated with total and all compositional PVs (all p0.05) (Table, Figure). Conclusion The overall burden of coronary atherosclerosis is associated with AVC at baseline. However, the progression of AVC is associated only with the progression of calcified PV but not with that of total and non-calcified PV. Representative case Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): The National Research Foundation (NRF) of Korea funded by the Ministry of Science and ICT (MSIT)

Background/Purpose In aortic valve stenosis calcification begins with nucleation on extracellular vesicles. In order to study early-stage disease, validated animal models are needed. The Drolet rabbit model is relevant due to tricuspid valve, but failed to consistently produce stenosis probably due to regimen administration. We compared a modified rabbit model and investigated the mechanisms and patterns of calcification. Methods New Zealand rabbits introduced to normal chaw+1% cholesterol+8750 IUs Vitamin D2/kg (Sigma) daily, in olive oil given in a bisquit vs control animals, for 8 weeks. Aortic valve area (AVA) and mean gradient (meanGr) was assessed with echocardiography (Vivid 7, M3S transducer, GE). At 8 weeks animals were sacrificed and valves were snap-frozen to −80°C. From each animal, one cusp was analyzed with Fourier-Transformed Infrared Spectroscopy (FT-IR, Nicolet 6700 spectrometer, OMNIC 7.3 software), another cusp was processed in alcoholic solution and the third was fixed 0.5 μm thin on 4% PFA; supernatant and tissue respectively examined with multispectral optical imaging. Valves from patients with severe stenosis were used for qualitative comparisons. Results At 8 weeks versus baseline, AVA reduced (0.5 cm2 to 0.3 cm2) and meanGr increased (1.1 to 2.95 mmHg, p Conclusions The modified high-fat-vitamin D2 rabbit model produces aortic valve stenosis, with chemically identical mineralization to human lesion. Multispectral photonics demonstrate the presence of calcified membranous extracellular particles, a hallmark of cardiovascular calcification. Dendrimer-like depositions correspond to growing deposits. The model is suitable as a research platform purposed for aortic valve stenosis. Figure 1. A: Image from alcoholic solution supernatant. The bright spots have high conductivity due to Ca 2+ deposition. B: ImageJ surface plot of circulated region confirms calcification. C: 3D-plot illustrates mineralization of membranes. D: 3D-plot of human aortic valve. E: Hypermicroscopic image of rabbit valve tissue: dendrimer-like and mineral cation deposits. Funding Acknowledgement Type of funding source: Public Institution(s). Main funding source(s): National and Kapodistrian University of Athens, Greece; Concordia University, Montreal, Canada

Background Stromal cell-derived factor-1 (SDF-1 or CXCL12) and its receptors CXCR4/CXCR7 have prominent role in cardiovascular development and myocardial repair following ischemic injury. Nevertheless, detailed mechanisms of the cell specific role of SDF-1 are poorly understood. Since SDF-1-EGFP lineage tracking revealed high expression of SDF-1 in smooth muscle cells, we aimed to investigate the cell specific role by generating a smooth muscle cell specific SDF-1 (SM-SDF-1−/−) knockout mouse model. Methods SDF-1 expression was analyzed utilizing SDF-1-EGFP reporter mice. Conditional SM-SDF-1 KO mice were generated using Tagln-Cre; SDF-1fl/fl mice. Hearts were analysed with histology and high-resolution episcopic microscopy. Cardiac function was assessed utilizing echocardiography. RNAseq, qRT-PCR, flow cytometry and western blotting were performed. Cardiac fibrosis, apoptotic index, cell proliferation, aortic valve calcification were analyzed. SM-SDF-1−/− mice were treated with the CXCR7 agonist TC14012 (10mg/kg/I.P). Results SDF-1-EGFP lineage tracking and immunofluorescence revealed high expression of SDF-1 particularly in smooth muscle cells and less frequently in perivascular and endothelial cells. Conditional SM-SDF-1−/− mice showed a high pre- and perinatal mortality (50%). Immunohistochemistry of SM-SDF-1−/− mice revealed severe cardiac hypertrophy, associated with increased cardiac fibrosis, apoptotic cell death, thinned and dilated arteries and significantly decreased M2 like CD11b+/CD206+ cells. Echocardiography confirmed concentric hypertrophy, with decreased stroke volume. As a possible reason for cardiac hypertrophy, SDF-1 mutants exhibited aortic stenosis due to aortic valve thickening associated with downregulation of the SDF-1 co-receptor CXCR7. We further noticed increased plasma levels of SDF-1 in aortic stenosis patients suggesting a cardioprotective role. Transcriptome analyses from KO hearts showed an abnormal extracellular matrix (ECM) remodelling with a specific upregulation of the important valve related proteoglycans Versican, Glycan. Western blot analysis revealed activation of AKT and ERK, whereas CXCR7 expression was significantly downregulated in KO mice. To rescue the phenotype we treated KO mice with the CXCR7 agonist (TC14012) which partially attenuated aortic valve remodelling through activation of the ERK signalling pathway. Conclusion Our data suggest that SDF-1 is critically involved in maintaining the homeostasis of the aortic valve by regulating CXCR7 signalling. Pharmacological activation of CXCR7 might be a promising therapeutic target to limit the progression of aortic valve stenosis. Ghadge_SM-SDF-1−/− Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Austrian Science Fund, Austrian research promotion agency

Background Calcific aortic valve disease (CAVD) is the most common heart valve disease among adults. It is a progressive disease whose final step leads to severe aortic stenosis (AS). Pharmacotherapeutic strategies aimed to limit the progression of valve leaflet calcification could be beneficial to slow-down the CAVD progression and to preserve left ventricular function. Several recent clinical studies demonstrated that lower serum magnesium (Mg) level is associated with vascular calcification. Whether serum magnesium is a determinant of aortic calcific stenosis progression remains unkwown. Methods In an ongoing prospective cohort of AS patients (COFRASA/GENERAC) we studied the association between serum Mg with the aortic valve calcification prevalence and progression. Serum Mg was measured at baseline in both its ionized (iMg) and total (tMg) forms. AS stenosis severity was evaluated at baseline and yearly thereafter using mean pressure gradient (MPG), the aortic valve area indexed to body surface (AVAi) assessed by echocardiography and the degree of aortic valve calcification (AVC) assessed by computed tomography. Annual progression was calculated as: (final measurement − baseline measurement)/follow-up duration. Results We enrolled 356 patients (73.1±10 years, 73% men), the mean follow-up duration was 2.5±2 years. There was a highly significant correlation between iMg and t Mg concentrations values (r=0.85, p0,45 mmol/L (1.78±0.94). Annual Mean MGP and AVAi also progressed greater in patients with low iMg but without reaching a significant level. Similar association was not found with tMg. In multivariate analysis, iMg remained significantly associated with the progression of AVC (odds ratio per 0.1 mmol/L increment [95% confidence interval] = 0.36 [0.15–0.83]; p=0.015) independently of age, tMg, glucose, type 2 diabetes, Tobacco use, baseline AVC, MPG and AVAi. Conclusion In a prospective cohort of asymptomatic patients with a wide range of AS severity, low serum ionized Mg but not low total Mg was independently associated with AVC progression. Acknowledgement/Funding ANR -RHU-STOPAS

The aim of this study was to determine whether CS, measured on the preoperative contrast enhanced multislice computed tomography (MSCT), is associated with Device Success (DS), Major Adverse Cardiac Events (MACE) and paravalvular leaks (PVL) after TAVR. Methods We included 352 consecutive patients who underwent TAVR with a preoperative standardized contrast enhanced MSCT. Valvular calcifications detection was defined by adding +100 Hounsfield Unit (HU) to mean HU determined by a Region Of Interest placed in the contrast enhanced ascending aorta. CS was then indexed to the aortic annulus surface (CSi). Endpoints were DS and 30-days MACE according to Valve Academic Research Consortium-2 consensus document, and PVL greater than or equal to grade 2. Results DS was obtained for 305 patients. In multivariate analysis, CS and CSi were negatively and independently associated with DS: OR=0.88, 95% CI 0.81–0.96, p=0.004; and OR=0.94, 95% CI 0.91–0.98, p=0.002 respectively. There was no association between MACE and CS (p=0,953) and CSi (p=0,757). PVL was positively associated with CS (p Multivariate analysis for device success Multivariate analysis for device success procedure Initial Model Final Model OR (95% CI) p OR (95% CI) p Age (years) 1.06 (1–1.12) 0.058 Size (cm) 1.02 (0.98–1.07) 0.26 Hypertension 0.35 (0.13–0.92) 0.033 0.34 (0.13–0.87) 0.024 Diabete mellitus 1.72 (0.68–4.34) 0.249 Mean gradient 0.99 (0.97–1.02) 0.517 Mitral regurgitation 0.6 (0.28–1.27) 0.18 Valve size ≤23 1 (REF) 26 1.85 (0.8–4.27) 0.152 29 4.08 (1.32–12.59) 0.015 4.72 (1.62–13.78) 0.004 31 1.42 (0.32–6.38) 0.647 CS (for an increase of 1000 pts) 0.89 (0.81–0.98) 0.017 0.88 (0.81–0.96) 0.004 CSi (for an increase of 100 pts) 0.94 (0.9–0.99) 0.013 0.96 (0.91–0.98) 0.002 Example of CS measurement Conclusion In TAVR, the aortic valvular CS, measured on preoperative contrast enhanced MSCT, is significantly associated with DS and PVL, but not with 30-days MACE. Its routine use could be relevant to appreciate success chances of TAVR.

Background Aortic stenosis (AS) is the most common acquired valvular heart disease. Calcification of the aortic valve (AV) cusps is the main pathogenetic mechanism of AS formation, however triggering and progression mechanisms of it are not fully understood. Aortic valve interstitial cells (AVIC) are one of the main cell populations responsible for the AV structure and homeostasis. The aim of the study was to characterize the ability of AVIC to osteogenic differentiation using the model of the primary culture of AVIC of patients with aortic stenosis and assess the expression of genes involved in osteogenesis (RUNX2, BMPG2, SPRY1, SOX9, CTNNB1, POSTN, OPG and OPN). Materials and methods The study was carried out on primary human AVIC culture obtained by enzymatic dissociation of experimental valve specimens from patients with AS (n=15) and the control specimens from the orthotopic heart transplantation recipients (n=10). To assess the osteogenic potential of AVIC, the routine stem cell osteodifferentiation protocol based on the culture medium with addition of inductors (10 mM β-glycerophosphate, 0.1 μM dexamethasone, and 50 μg/ml ascorbic acid) was used. Calcium deposits were demonstrated by Alizarin Red staining. Analysis of the expression of osteogenic differentiation genes, such as RUNX2 (runtrelated transcription factor 2), BMP2 (bone morphogenetic protein 2), SPRY1 (Sprouty RTK signaling antagonist 1), SOX-9 (SRY-box9), CTNNB1 (β-catenin1), POSTN (periostin), OPG (osteoprotegerin), and OPN (osteopontin), was performed by real-time PCR. Results The inductors of osteogenic differentiation provoked greater mineralization of AVIC cultures derived from the patients with AS than that observed in control group (p=0.0003). The expression of RUNX2 and SPRY1 in non-differentiated cells was reduced compared with control cells in patients with bicuspid AV (p=0.02). After 21 days of the osteogenic induction, the expression of RUNX2 and SPRY1 increased in all three groups. At the same time, the expression of SPRY1 was lower in the group with bicuspid AV compared with tricuspid AV (p=0.007). The expression level of BMP2 did not differ between groups in unstimulated AVIC, however, it increased after osteogenic differentiation in the group of patients with tricuspid AV (p=0.017). OPN expression was higher in cells from tricuspid AV, while OPG expression was reduced in patients with both bicuspid and tricuspid AV (p Conclusions The osteogenic potential of AVIC is increased in patients with aortic stenosis. The gene expression profile of osteogenic differentiation differs in patients with a bicuspid and tricuspid aortic valve. Damage of protective mechanisms may be a potential mechanism for accelerated valve calcification mostly in patients with tricuspid aortic valve. Acknowledgement/Funding Russian Foundation for Basic Research, project 18-015-00016

Background Aortic valve calcification associated with either bicuspid or tricuspid aortic valve remains among the most dangerous cardiovascular diseases but cellular and molecular reasons of this pathology are not fully understood. It has been shown that NOTCH1 haploinsufficiency in endothelial cells derived from iPS and also inhibition of NOTCH1 in aortic valve endothelial cells gives rise to the activation of proosteogenic pathways. Endothelial cells regulate underlying interstitial cells and govern them to differentiation. Thus, elucidation of molecular mechanisms of endothelial dysfunction could shed light on valve calcification pathogenesis. Purpose To elucidate key genes responsible for valve endothelial dysfunction in aortic valve calcification. Methods Primary human aortic valve endothelial cells (VEC) were isolated from 45 patients (BAV, n=21; TAV, n=26) with calcific aortic valve disease and from 8 healthy donors. Expression of corresponding genes was estimated by qPCR. Results We checked expression of about 30 genes related to Notch/Wnt/Bmp pathways and shear stress response. Endothelial cells of patients with calcification had significantly lower mRNA levels of Notch component genes NOTCH2, NOTCH3, DLL4 and JAG1 as well as of Notch target genes HEY1, SNAIL and SLUG. Expression of Wnt-related genes such as DKK1, GREM1 and TCF4 was altered in VEC from calcified valves. Interestingly, mRNA level of ROBO4, recently suggested to be involved in endothelial dysfunctions, was significantly higher in VEC of patients with calcification. Moreover, endothelium from calcified valves had impaired expression of matrix gla protein (MGP), which is known to sequester calcium and inhibit soft tissue calcification in the vasculature and aortic valve. Conclusions Our data show that Notch pathway genes are downregulated in valve endothelial cells of the patients with aortic valve calcification. Our results suggest also implication of shear stress responsive genes and genes of endothelial performance in changing the properties of valve endothelial population of calcified valves and thus presumably to the pathogenesis of aortic valve calcification. Acknowledgement/Funding Russian Foundation For Basic Research 18-34-00277

Background Calcific aortic valve disease is the 2nd most frequent cause of open heart surgery. The valve interstitial cells (VIC) are crucial for calcification. SNF472 (a derivative of phytic acid) is a calcification inhibitor currently in clinical development for the treatment of cardiovascular calcification (Phase 2 CaLIPSO trial, EudraCT 2016–002834–59). SNF472 has been shown to inhibit vascular calcification in several preclinical models. Purpose 1. Establish a new model of calcification in cultured human VIC; 2. Investigate whether SNF472 would inhibit calcification in this model, and 3. Study if SNF472 might inhibit ongoing calcification processes. Methods Healthy and calcified aortic valves were obtained from heart transplant recipients and patients undergoing aortic valve replacement due to calcific valve disease, respectively. VIC were isolated and seeded in basic growth medium, osteogenic differentiation medium (Osteodiff) alone, and with addition of different concentrations of SNF472. The following series of studies were performed: 1. VIC from healthy and calcified valves were cultured for three weeks with Osteodiff; 2. VIC from calcified valves were cultured for 3 weeks in Osteodiff media with 0, 1, 3, 10, 30, or 100 μM SNF472; 3. VIC from calcified valves were cultured for 3 weeks in Osteodiff media in total, but after 1 or 2 weeks 30 or 100 μM SNF472 was added to the cultures (n=8). Calcification was visualized by Alzarin Red staining and quantified by spectrophotometry. Statistics analysis was performed nonparametric One-Way ANOVA (Friedman and Kruskal–Wallis tests) with Dunn's post-test. Results Calcification was found to be 30% stronger in cultures of VIC from calcified valves as compared to cultured VIC from healthy valves (p=0.03). SNF472 successfully inhibited VIC calcification in a dose-dependent manner. SNF472 concentrations of 1, and 3 μM inhibited calcification by 7% (not significant) and 66% (p=0.08) respectively. Concentrations of 10, 30, and 100 μM completely inhibited calcification. 30 and 100 μM of SNF472 added after 1 week reduced ongoing calcification by 84% (p Conclusions VIC from calcified valves have a more pro-calcification phenotype than VIC from healthy valves. SNF472 is able to inhibit the development VIC calcification in vitro. By early intervention SNF472 is also able to stop the progression of ongoing calcification. SNF472 shows to be a promising therapy to treat heart valve calcification. Acknowledgement/Funding EC FP7 (GA 609020), Balearic Islands Government grant (ES01/TCAI/41_2017), FEDER 2014-2020, Laboratoris Sanifit, Palma, Spain; University of Oslo

Introduction Calcific aortic valve disease (CAVD) is one of the most common heart diseases worldwide, and its prevalence has increased in developed countries along with aging populations. Subclinical CAVD is characterized by aortic valve calcification (AVC) that may lead to aortic stenosis. AVC shares the same risk factors with atherosclerotic disease. Recently, nuclear magnetic resonance (NMR)-based lipoprotein particles, low-density and high-density lipoprotein particles (LDL-p, HDL-p) in particular, have emerged as novel markers of atherosclerotic disease. It has been unclear, however, whether NMR-based particles are associated with AVC. Purpose To examine cross-sectional association of NMR-based lipoprotein particles, standard lipids with AVC, and compare their strengths of association. Methods We studied a community-based random sample of Japanese men aged 40–79 years examined in 2006–08 in Japan. We excluded participants with history of myocardial infarction, stroke, aortic valve surgery, serum triglyceride concentration ≥400 mg/dl, estimated glomerular filtration rate (eGFR) 0. Lipoprotein particles were measured using NMR spectroscopy. In our main analysis, we obtained multivariable-adjusted odds ratios (ORs) and 95% confidence intervals (95% CI) of prevalent AVC in the top quartile (Q4) for each lipid index in reference to the lowest quartile (Q1). Adjusting covariates include age, smoking, drinking, systolic blood pressure, glycated hemoglobin, medications for hypertension and/or for diabetes, eGFR, C-reactive protein. For sensitivity analysis, we repeated similar analyses presenting ORs per 1 standard deviation (SD) higher NMR-lipoproteins particles or standard lipids. Results Of 874 participants we analyzed, 153 (17.5%) men had AVC. The average (SD) age was 63.3 (10.0) years. Those with AVC tended to be older, likely to have clustering of atherosclerotic risk factors. Multivariable-adjusted ORs of prevalent AVC for Q4 versus Q1 were significantly positive for LDL-p [OR, 2.21 (95% CI: 1.24, 3.95] and low-density lipoprotein cholesterol (LDL-c) [2.17 (1.24, 3.80)]. In contrast, neither HDL-p nor high-density lipoprotein cholesterol (HDL-c) was significantly associated with AVC. In sensitivity analyses, however, the corresponding ORs per 1SD higher lipids/lipoprotein particles were smaller in LDL-p [1.33 (1.09, 1.63)] than LDL-c [1.41 (1.15, 1.73)]. The association of HDL-p became significant [1.28 (1.02, 1.61)] (per 1SD decrement) in the sensitivity analysis. Conclusion The overall associations of AVC with NMR-based lipoprotein particles (i.e. LDL-p, HDL-p) are comparable to those of standard lipids. This finding is parallel to the association between atherosclerotic cardiovascular diseases with NMR-based lipoproteins.

Background An individual with bicuspid aortic valve (BAV) has a higher risk of developing aortic valve calcification (AVC) than people with tricuspid aortic valve. AVC in patients with BAV and TAV is known to be distinct in molecular level. But the epigenetic mechanism of AVC in patients with BAV is still undefined. Purpose To describe the role of microRNA (miRNA) in the AVC process of patients with BAV Methods We collected aortic valve samples of 5 patients with BAV, of which 2 have AVC and 3 of which have not. We performed RNA-seq technology on an Illumina Hiseq 2500/2000 platform to characterized all feathers of miRNAs in BAV. We used miRBase20.0 to look for known miRNA. Gene expression levels between groups of valves were compared with degeR to find out differential miRNAs. Then we predicated the target gene of miRNA by targetscan database. We performed the GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis for the target gene candidates of differential miRNAs to predict the potential function of AVC specific miRNAs. A value of P Results Compared with noncalcified aortic valves. We found that 33 miRNAs were upregulated and 29 are significantly downregulated in calcified aortic valves. The analysis showed the total of 62 miRNAs to be significantly altered as shown in the heat map in Figure1. the target gene candidates of differential miRNAs were enriched in numerous Go terms including the following: binding, protein binding, ion binding, catalytic activity, localization. The significantly (P Table 1 Pathway term Gene number P valvue Lysosome 70 0.001945 Axon guidance 66 0.013212 Glycerophospholipid metabolism 50 0.016075 Calcium signaling pathway 84 0.037645 Synaptic vesicle cycle 34 0.043983 Pyrimidine metabolism 52 0.04501 Homologous recombination 18 0.0453 Other glycan degradation 13 0.048068 Pathway analysis based on the KEGG database. Figure 1 Conclusion The present study proved that miRNAs play an important role in AVC of patients with BAV. The result may improve understanding of the special molecular alterations in AVC with BAV and may provide potential targets for future clinical applications. Acknowledgement/Funding the National Natural Science Foundation of China (No.81370435; 91339103; 81700405)

Calcific aortic valve stenosis (CAVS) is common in the ageing population and set to become an increasing economic and health burden. Once present, it inevitably progresses and has a poor prognosis in symptomatic patients. No medical therapies are proven to be effective in holding or reducing disease progression. Therefore, aortic valve replacement remains the only available treatment option. Improved knowledge of the mechanisms underlying disease progression has provided us with insights that CAVS is not a passive disease. Rather, CAVS is regulated by numerous mechanisms with a key role for calcification. Aortic valve calcification (AVC) is actively regulated involving cellular and humoral factors that may offer targets for diagnosis and intervention. The discovery that the vitamin K-dependent proteins are involved in the inhibition of AVC has boosted our mechanistic understanding of this process and has opened up novel avenues in disease exploration. This review discusses processes involved in CAVS progression, with an emphasis on recent insights into calcification, methods for imaging calcification activity, and potential therapeutic options.

Aims Aortic valve stenosis (AS) is an actively regulated process like atherosclerosis, which is accompanied by changes e.g. in endothelin-related genes. However, the role of endothelin peptides in AS is unknown. Methods and results We characterized the expression of the endothelin system in aortic valves of patients with normal valves ( n = 12), regurgitation, and fibrosis ( n = 6) and AS ( n = 18) by reverse-transcriptase–polymerase chain reaction and immunohistochemistry. The number of endothelin-1 (ET-1) positive cells was higher in AS than in control valves, while levels of ET-1 mRNA did not differ between groups. Endothelin receptor-A (ETA) mRNA levels were upregulated in stenotic valves (4.3-fold, P = 0.032) associated with a remarkable increase in number of ETA-immunopositive cells. ETB-receptor mRNA levels did not change during disease progression. Endothelin-converting enzyme-1 (ECE-1) mRNA levels were 42% lower ( P = 0.007) in stenotic valves. Finally, because ET-1 and ECE-1 have binding site for activator protein-1 (AP-1), we measured AP-1 DNA binding by gel shift assays, which showed significantly lower (76%, P = 0.003) activity in AS. Conclusion AS is characterized by distinct upregulation of ET-1 and its target receptor ETA, promoting growth, inflammation, and fibrosis. These findings suggest therapeutic potential for ETA-receptor antagonists in aortic valve calcification.

Aims Mitral annular calcification (MAC) is characterized by calcium and lipid deposition in the annular fibrosa of the mitral valve. Although individuals with MAC are at increased risk of cardiovascular events, little is known about the significance of this finding in patients with concurrent aortic stenosis (AS). The aim of this study was to describe the association of baseline MAC and aortic valve morphology in asymptomatic patients enrolled in the ASTRONOMER study, a multicentre study to assess the effect of Rosuvastatin on the progression of AS. Methods and results At baseline, transthoracic echocardiography was performed with two-dimensional and Doppler imaging following a standardized protocol. Echo measurements including left ventricular (LV) dimensions and aortic root dimensions were obtained according to the ASE recommendations. MAC was identified by bright echoes at the base of the mitral leaflets or annulus on 2D imaging, and aortic valve calcification by visualization of bright echoes on the aortic valve leaflets. The degree of calcification was semi-quantitated from absent to severe. The study population included 219 patients (57 ± 14 years; 129 males), divided into two pre-specified categories; bicuspid ( n = 133) and tricuspid ( n = 86) aortic valves. Baseline LV dimensions, aortic valve haemodynamics, and cholesterol profiles were similar between the two groups at baseline. Individuals with tricuspid aortic valves were older, more hypertensive, with higher degrees of MAC and AV calcification ( P < 0.001). The higher degree of MAC persisted in patients with tricuspid AV after adjustment for age and systolic blood pressure ( P = 0.004). Conclusion In patients with asymptomatic mild to moderate AS, MAC is more prevalent in those individuals with tricuspid AV, independent of age, and systolic blood pressure. Whether the degree of MAC may be a surrogate for atherosclerosis, and predict the subset of patients who will respond to statin therapy in preventing the progression of AS, remains to be determined.

Aims Similar risk factors and mediators are involved in calcific aortic stenosis (CAS) and atherosclerosis. Since normal valves harbour a low percentage of smooth muscle cells (SMCs), we hypothesize that the SMC phenotype participates in the pathogenesis of CAS. Method and results We analysed 12 normal and 22 calcified aortic valves for SMC markers and the expression of co-activators of SMC gene expression, myocardin and myocardin-related transcription factors (MRTF-A/B). Transforming growth factor β (TGFβ1) was used to upregulate SMC markers and co-activators in valve interstitial cells (VICs) and transmission electron microscopy (TEM) was used to detect the presence of SMC in atypical regions of the valve leaflets. Smooth muscle cell markers and co-activators, myocardin, MRTF-A, and MRTF-B, demonstrated an increased incidence and aberrant expression around calcified nodules in all 22 calcified valves as well as in surface and microvessel endothelial cells. Smooth muscle cell markers and MRTF-A were significantly increased in calcified valves. Transforming growth factor β1 (TGFβ1) (10 ng/mL) was able to significantly upregulate the expression of some SMC markers and MRTF-A in VICs. Transmission electron microscopy of the fibrosa layer of calcified valves demonstrated the presence of bundles of SMCs and smooth muscle-derived foam cells. Conclusion Smooth muscle cell markers and co-activators, myocardin and MRTFs, were aberrantly expressed in calcified valves. Transforming growth factor β1 was able to significantly upregulate SMC markers and MRTF-A in VICs. Transmission electron microscopy unequivocally identified the presence of SMCs in calcified regions of valve leaflets. These findings provide evidence that the SMC phenotype plays a role in the development of CAS.

This editorial refers to ‘Aortic stenosis, atherosclerosis, and skeletal bone: is there a common link with calcification and inflammation?’[†][1], by M.R. Dweck et al. , on page 1567 I had … . come to an entirely erroneous conclusion which shows, my dear Watson, how dangerous it is to reason from insufficient data. Sherlock Holmes to Dr Watson in ‘The Adventure of the Speckled Band’, 1892.1 Calcific aortic stenosis (AS) was initially characterized as a degenerative process (disorder) because of a tendency to describe any disorder that was not well understood and often occurred in the ‘elderly’ as degenerative. In the last ‘15–20’ years, the observation of the presence of risk factors for arterial atherosclerosis in patients with calcific AS, the production of aortic valve calcification in animals fed grossly high levels of lipids, the presence of an osteoblast phenotype in calcific AS, and many studies showing features of and similarities to arterial atherosclerosis led to a … [1]: #fn-2

Aims The pathophysiology of aortic stenosis shares many similarities with atherosclerosis and skeletal bone formation. Using non-invasive imaging, we compared aortic valve calcification and inflammation activity with that measured in atherosclerosis and bone. Methods and results Positron emission and computed tomography was performed using 18F-sodium fluoride (18F-NaF, calcification) and 18F-fluorodeoxyglucose (18F-FDG, inflammation) in 101 patients with calcific aortic valve disease (81 aortic stenosis and 20 aortic sclerosis). Calcium scores and positron emission tomography tracer activity (tissue-to-background ratio; TBR) were measured in the aortic valve, coronary arteries, thoracic aorta, and bone. Over 90% of the cohort had coexistent calcific atheroma, yet correlations between calcium scores were weak or absent (valve vs. aorta r 2 = 0.015, P = 0.222; valve vs. coronaries r 2 = 0.039, P = 0.049) as were associations between calcium scores and bone mineral density (BMD vs. valve r 2 = 0.000, P = 0.766; vs. aorta r 2 = 0.052, P = 0.025; vs. coronaries r 2 = 0.016, P = 0.210). 18F-NaF activity in the valve was 28% higher than in the aorta (TBR: 2.66 ± 0.84 vs . 2.11 ± 0.31, respectively, P < 0.001) and correlated more strongly with the severity of aortic stenosis ( r 2 = 0.419, P < 0.001) than 18F-NaF activity outwith the valve (valve vs. aorta r 2 = 0.167, P < 0.001; valve vs. coronary arteries r 2 = 0.174, P < 0.001; valve vs. bone r 2 = 0.001, P = 0.806). In contrast, 18F-FDG activity was lower in the aortic valve than the aortic atheroma (TBR: 1.56 ± 0.21 vs . 1.81 ± 0.24, respectively, P < 0.001) and more closely associated with uptake outwith the valve (valve vs. aorta r 2 = 0.327, P < 0.001). Conclusion In patients with aortic stenosis, disease activity appears to be determined by local calcific processes within the valve that are distinct from atherosclerosis and skeletal bone metabolism. Trial Registration: ClinicalTrials.gov number: [NCT01358513][1] [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01358513&atom=%2Fehj%2F34%2F21%2F1567.atom

Aims Westernized countries face a growing burden of cardiovascular calcification and osteoporosis. Despite its vast clinical significance, the precise nature of this reciprocal relationship remains obscure. We hypothesize that cardiovascular calcification progresses with inflammation and inversely correlates with bone tissue mineral density (TMD). Methods and results Arterial, valvular, and bone metabolism were visualized using near-infrared fluorescence (NIRF) molecular imaging agents, targeting macrophages and osteogenesis. We detected significant arterial and aortic valve calcification in apoE−/− mice with or without chronic renal disease (CRD, 30 weeks old; n = 28), correlating with the severity of atherosclerosis. We demonstrated decreases in osteogenic activity in the femurs of apoE−/− mice when compared with WT mice, which was further reduced with CRD. Three-dimensional micro-computed tomography imaging of the cortical and cancellous regions of femurs quantified structural remodelling and reductions in TMD in apoE−/− and CRD apoE−/− mice. We established significant correlations between arterial and valvular calcification and loss of TMD ( R 2 = 0.67 and 0.71, respectively). Finally, we performed macrophage-targeted molecular imaging to explore a link between inflammation and osteoporosis in vivo . Although macrophage burden, visualized as uptake of NIRF-conjugated iron nanoparticles, was directly related to the degree of arterial and valvular inflammation and calcification, the same method inversely correlated inflammation with TMD ( R 2 = 0.73; 0.83; 0.75, respectively). Conclusion This study provides direct in vivo evidence that in arteries and aortic valves, macrophage burden and calcification associate with each other, whereas inflammation inversely correlates with bone mineralization. Thus, understanding inflammatory signalling mechanisms may offer insight into selective abrogation of divergent calcific phenomena.

Aims Fetuin-A has been identified as a potent circulating inhibitor of ectopic calcification. We investigated the relationship between baseline fetuin-A serum levels and the rate of progression of aortic valve calcification (AVC) in non-dialyzed patients with aortic valve disease (AVD). Methods and results Seventy-seven patients (mean age 70 ± 8 years) with echocardiographically proven AVD were collected. In all patients, serum fetuin-A levels, creatinine, calcium, lipid parameters, and C-reactive protein were measured at baseline. For quantification of AVC progression, all patients underwent multislice spiral computed tomography examinations at baseline and after a mean follow-up of 12.6 ± 1.4 months (range 7–18 months). In a multifactorial analysis of covariance including fetuin-A levels, baseline AVC score, the covariables sex, age, body mass index, C-reactive protein, glomerular filtration rate, serum lipids, diabetes, smoking status, and hypertension, only serum fetuin-A levels significantly predict the progression of AVC ( P < 0.001). Post hoc analysis demonstrated that patients with baseline fetuin-A levels lower than the median of the cohort (0.72 g/L) showed a significantly higher increase of AVC scores (34.6 ± 31.4%) than patients with fetuin-A levels larger than the median (10.0 ± 11.2%, P < 0.001) despite comparable baseline AVC scores. In addition, fetuin-A levels were associated with major adverse clinical events (MACE; P = 0.03). Conclusion Serum levels of the calcification inhibitor fetuin-A are associated with the progression of AVC and MACE, independent of the renal function and inflammation.

Aortic valve stenosis is a potentially serious condition. Progression from mild to severe aortic stenosis is well-recognized but there are few data as to the likely rate of progression. Clinical outcome and cardiac catheterization data were reviewed for 65 patients with valvar aortic stenosis. Each patient had been investigated by cardiac catheterization on at least two occasions, the interval between studies ranging between 1 and 17 years (mean 7 years). In 60 cases the aortic valve gradient had increased, from a median of 10 mmHg (range 0-60) to a median of 52 mmHg (range 15-120). The mean rate of increase of gradient was 6.5 mmHg per year, and was significantly faster in patients in whom there was aortic valve calcification or aortic regurgitation present at the first catheter study (P less than 0.02). This study shows that progression of aortic stenosis may be very rapid, and correlates with valve calcification and regurgitation. If cardiac surgery is proposed for co-existing coronary or mitral valve disease in patients with mild or moderate aortic valve gradients, then aortic valve replacement should be considered at that time.

AIMS Endothelial destruction and calcification primarily occur at the aortic side of the calcified aortic valves (AVs). This study investigated whether degenerative AV stenosis (AS) is associated with the presence of valvular endothelial senescence and a reduction in the number and function of endothelial progenitor cells (EPCs). METHODS AND RESULTS Fifteen patients with severe AS and 18 age-matched control subjects were enrolled. Senescence-associated beta-galactosidase activity was mostly localized on the valvular endothelial cells (ECs) of the explanted AVs and highly coincided with the calcified lesion at the aortic side. The number (9.3 +/- 8.3 vs. 20.5 +/- 9.0 cells per 10(6) mononuclear cells; P < 0.01) and the migratory capacity (107.8 +/- 54.6 vs. 185.0 +/- 68.8 cells per 1000 cells; P < 0.01) of EPCs evaluated by FACS analysis or migration assay were significantly reduced in AS when compared with control. As possible mechanisms of alterations in EPCs, caspase-3 activity was significantly increased, whereas telomere-repeating factor-2 expression quantified by western blot was significantly reduced in EPCs from AS when compared with control. CONCLUSION Reduced regenerative capacity of valvular ECs due to senescence and decreased levels of EPCs might be, at least in part, a pathological link for the destruction of valvular ECs, resulting in progression of degenerative AS.

Calcific aortic stenosis, with a prevalence of 3-9%, is the most frequent heart valve disease and the main cause for valve replacement in patients over 60 years of age. Once thought to be caused by a passive calcium precipitate within the aortic valve leaflets, there is now increasing evidence that development and progression of calcific aortic valve disease may be triggered by underlying genetic and cardiovascular risk factors, and is regulated by an active cellular process involving inflammatory pathways. Targeted drug therapy to prevent the progression of calcific aortic valve disease should ideally be based on the knowledge of risk factors and the molecular pathogenesis of the disease. Conflicting data exists on the potency of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (i.e. statins) to influence both risk factors and inflammatory pathways by lowering lipid levels and exerting anti-inflammatory properties, respectively. In this review, various aspects of the molecular pathogenesis of calcific aortic stenosis will be summarized and connected with recent experimental and clinical studies that address the potential benefit of the targeted drug therapy by statins in order to prevent the progression of the disease.

AIMS To define the natural history and predictors of outcome in mild and moderate aortic stenosis (AS). METHODS AND RESULTS One hundred and seventy-six consecutive asymptomatic patients (73 women, age 58+/-19 years) with mild to moderate AS (jet velocity 2.5 to 3.9m/s) were followed for 48+/-19 months. Haemodynamic progression and clinical outcome was analysed. Event-free survival with end-points defined as death (n=34) or aortic valve surgery (n=33), was 95+/-2%, 75+/-3% and 60+/-5% at 1, 3 and 5 years, respectively. Both, cardiac and non-cardiac mortality were significantly increased, resulting in a 1.8 times higher mortality than expected (P

Aims The study evaluated the relationship between cardiovascular risk factors (CRF), gene polymorphism, calcification and fibrosis of stenotic aortic valves. Methods and results The calcium content of 187 excised stenotic aortic valves was determined using atomic absorption spectroscopy. Hydroxyproline content was quantified. Left-heart catheterization was performed. CRF and genotypes of the interleukin 10, connective tissue growth factor (CTGF) and chemokine receptor 5 (CCR5) polymorphisms were assessed. Calcification consisted of Ca-deficient hexagonal hydroxyapatite, Ca10− x (HPO4) x (PO4)6− x (OH)2− x ; with \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(0{\leqslant}x{\leqslant}1\) \end{document}. Calcification (quintiles) was positively associated with the mean gradient across the aortic valve (44±14, 52±17, 54±16, 60±15, 68±19 mmHg; \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(p{

Purpose: Optimal management of concomitant coronary artery disease (CAD) in patients undergoing transcatheter aortic valve implantation (TAVI) is contentious. This study aimed to assess the role of routine non rate-controlled CT coronary angiography (CTCA) in assessing CAD in patients referred for TAVI, thereby potentially precluding the need for invasive coronary angiography (ICA). CTCA in this cohort can be difficult due to the high burden of coronary calcium and the inability to perform standard pharmacological rate control. Methods: All patients underwent an ECG-gated 128-slice calcium score and CT angiogram to assess peripheral vasculature, aortic anatomy, aortic valve calcification and location of the coronary ostia. Coronary artery calcium scores (CACS) and CTCA images were scored by two independent, blinded observers and compared with ICA performed prior to TAVI. Significant coronary stenosis was defined as ≥70% and ICA was deemed the gold standard for true disease status. Sensitivity, specificity, positive (PPV) and negative predictive values (NPV) were calculated for CTCA at a patient level. Results: 70 consecutive patients referred for consideration of TAVI were studied: 40% male (n=28), mean age 78.9yrs (±7.91), 23% diabetic (n=16). Significant CAD was demonstrated in 30% of ICA studies (1 vessel disease (VD) 20% [n=14], 2VD 8.6% [n=6], 3VD 1.4% [n=1]). The sensitivity, specificity, PPV and NPV of CTCA for the detection of significant coronary stenoses were 57.9% (CI: 33.5% to 79.7%), 87.0% (CI: 75.1% to 94.6%), 61.1% (CI: 35.8% to 82.6%) and 85.4% (CI: 73.3% to 93.5%) respectively. The vast majority of false positive (5/6; 83%) and false negative (6/7; 86%) CTCAs were seen in patients in the highest quartile of CACS (>400). Conclusions: In patients undergoing CT assessment prior to TAVI, non rate-controlled CTCA shows poor sensitivity but good specificity and negative predictive value for the detection of significant CAD. A CAC score >400 may represent a threshold above which invasive angiography is mandated.

Background: Transesophageal echocardiography (TEE) is often considered as the reference method for the measurement of the aortic annulus diameter (AAD) during transcatheter aortic valve implantation (TAVI). In the present study, we evaluated whether Multislice Computed Tomography (MSCT) could reproduce and thus substitute to TEE. Method: We compared AAD measurements performed using TEE, MSCT and transthoracic echocardiography (TTE) in 129 consecutive patients with severe aortic stenosis (AS) referred for TAVI. Using MSCT, AAD was measured in the 3-chamber view and at the level of the virtual basal ring. We calculated the mean (MD) of the long-axis (LA) and short-axis (SA) diameters, mathematic transformations emphasizing the weight of the SA (MD2 to MD5, MD2 = 2SA + LA/3 ...), AAD derived from the cross-sectional area and from the circumference of the virtual basal ring. Comparisons between methods were assessed using single-measure intraclass correlation coefficient (ICC) and agreements as regard to the TAVI strategy (decision to implant and choice of the prosthesis' size based on manufacturer's cutoffs recommendations) expressed using the kappa value. Results: The 3C method (ICC=0.79, 95% interval 0.73-0.83), MD4 (ICC=0.76, 95% interval 0.69-0.81) and MD5 (ICC=0.75, 95% interval 0.67-0.81) provided the highest correlation and the best agreement to TEE (kappa value of 0.47, 0.27 and 031 respectively) but remained lower than TTE (ICC=0.87; 95% interval 0.83-0.91; kappa=0.66). The agreement between MSCT and TEE varied with the degree of eccentricity of the aortic annulus or the degree of aortic valve calcification but never reached the values observed with TTE. Conclusion: No direct or indirect MSCT method provided higher correlations to TEE than TTE. Consequently, no MSCT method could reproduce and thus substitute to TEE. Randomized prospective studies are clearly needed to evaluate which method provides the best clinical results, but we definitely demonstrate that MSCT and TEE are not interchangeable.

This study aimed at identifying factors influencing aortic valve calcification in old age. Echocardiographic and Doppler characteristics of the aortic valve were compared with possible clinical and biochemical predictors in 501 people aged 75-86 years and in 76 aged 55-71. Slight calcification was seen in 222 people (40%) and severe calcification in 72 (13%); 21 people had moderate or severe aortic stenosis. Age (P = 0.000) and serum parathyroid hormone (P = 0.015) were higher and body mass index lower (P = 0.002) in the presence of aortic valve calcification. In multivariate analysis, age (P = 0.000), hypertension (P = 0.005) and body mass index (P = 0.005) were independent predictors of aortic valve calcification, and age (P = 0.022) and serum ionized calcium (P = 0.037) of valve stenosis. The odds ratio (95% confidence interval) for valve calcification was 1.89 (1.42-2.50) for a 10-year increase in age, 1.74 (1.19-2.55) in the presence of hypertension, and 1.39 (1.10-1.76) for a 5 kg.m-2 decrease in body mass index. Sex, smoking, diabetes, serum lipids and insulin were unrelated to valvular calcification. These data suggest that leanness and a history of hypertension increase the likelihood of senile aortic valve calcification. Calcium metabolism may also be of significance. The mechanisms of these associations deserve further study.

The potential predictors of left ventricular mass in old age were studied in a random sample of people born in 1904, 1909, 1914 (n = 501) and 1920-35 (n = 76). Data on the left ventricle with mass calculation, quantitative data on valve disorders and biochemical data were collected. Left ventricular mass (corrected for height) was positively related to male gender, age, body mass index and systolic blood pressure. It was also significantly higher in the presence of aortic valve calcification, a low velocity ratio (indicating aortic valve obstruction), and aortic or mitral regurgitation. In men, left ventricular mass was higher in the presence of coronary artery disease and inversely related to serum high density lipoprotein cholesterol. In multivariate analysis, independent predictors of left ventricular mass without sex interaction were age (standardized coefficient beta = 0.23, P = 0.000), male gender (beta = 0.38, P = 0.000), body mass index (beta = 0.22, P = 0.000), systolic blood pressure (beta = 0.21, P = 0.000), velocity ratio (beta = -0.11, P = 0.010), detectable aortic regurgitation (beta = 0.11, P = 0.014) and moderate-to-severe mitral regurgitation (beta = 0.21, P = 0.000). Thus, the increase in left ventricular mass with age can largely be related to discrete predictors. The impact of valve disorders is substantial.