The angiotensin AT2-receptor agonist compound 21 is an antagonist for the thromboxane TP-receptor - Implications for preclinical studies and future clinical use.

Since the AT2-receptor (AT2R) agonist C21 has structural similarity to the AT1-receptor antagonists Irbesartan and Losartan, which are antagonists not only at the AT1R, but also at thromboxane TP-receptors, we tested the hypothesis that C21 has TP-receptor antagonistic properties as well. Isolated mouse mesenteric arteries from C57BL/6 J and AT2R-knockout mice (AT2R-/y) were mounted in wire myographs, contracted with either phenylephrine or the thromboxane A2 (TXA2) analogue U46619, and the relaxing effect of C21 (0.1 nM - 10 µM) was investigated. The effect of C21 on U46619-induced platelet aggregation was measured by an impedance aggregometer. Direct interaction of C21 with TP-receptors was determined by an ß-arrestin biosensor assay. C21 caused significant, concentration-dependent relaxations in phenylephrine- and U46619-contracted mesenteric arteries from C57BL/6 J mice. The relaxing effect of C21 was absent in phenylephrine-contracted arteries from AT2R-/y mice, whereas it was unchanged in U46619-contracted arteries from AT2R-/y mice. C21 inhibited U46619-stimulated aggregation of human platelets, which was not inhibited by the AT2R-antagonist PD123319. C21 reduced U46619-induced recruitment of ß-arrestin to human thromboxane TP-receptors with a calculated Ki of 3.74 µM. We conclude that in addition to AT2R-agonistic properties, C21 also acts as low-affinity TP-receptor antagonist, and that - depending on the constrictor - both mechanisms can be responsible for C21-induced vasorelaxation. Furthermore, by acting as a TP-receptor antagonist, C21 inhibits platelet aggregation. These findings are important for understanding potential off-target effects of C21 in the preclinical and clinical context and for the interpretation of C21-related myography data in assays with TXA2-analogues as constrictor.

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

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

Association of Left Atrial Size Measured by Non-Contrast Computed Tomography with Cardiovascular Risk Factors-The Danish Cardiovascular Screening Trial (DANCAVAS).

Left atrium (LA) size is associated with adverse cardiovascular events. The purpose of this study was to investigate the association of LA enlargement measured by non-contrast CT (NCCT) with traditional cardiovascular risk factors. Individuals aged 60-75 years from the population-based multicentre Danish Cardiovascular Screening (DANCAVAS) trial were included in this cross-sectional study. The LA was manually traced on the NCCT scans, and the largest cross-section area was indexed to body surface area. All traditional risk factors were recorded, and a subgroup received an echocardiographic examination. We enrolled 14,987 individuals. Participants with known cardiovascular disease or lacking measurements of LA size or body surface area were excluded, resulting in 10,902 men for the main analysis and 616 women for a sensitivity analysis. Adjusted multivariable analysis showed a significantly increased indexed LA size by increasing age and pulse pressure, while smoking, HbA1c, and total cholesterol were associated with decreased indexed LA size. The findings were confirmed in a supplementary analysis including left ventricle ejection fraction and mass. In this population-based cohort of elderly men, an association was found between age and pulse pressure and increasing LA size. Surprisingly, smoking, HbA1c, and total cholesterol were associated with a decrease in LA size. This indicates that the pathophysiology behind atrial cardiomyopathy is not only reflected by enlargement, but also shrinking.

Mitral Annulus Calcification and Cardiac Conduction Disturbances: A DANCAVAS Sub-study.

BACKGROUND: Due to its location very close to the bundle of His, mitral annulus calcification (MAC) might be associated with the development of atrioventricular (AV) conduction disturbances. This study assessed the association between MAC and AV conduction disturbances identified by cardiac implantable electronic device (CIED) use and electrocardiographic parameters. The association between MAC and traditional cardiovascular risk factors was also assessed. METHODS: This cross-sectional study analyzed 14,771 participants, predominantly men aged 60-75 years, from the population-based Danish Cardiovascular Screening trial. Traditional cardiovascular risk factors were obtained. Using cardiac non-contrast computed tomography imaging, MAC scores were measured using the Agatston method and divided into absent versus present and score categories. CIED implantation data were obtained from the Danish Pacemaker and Implantable Cardioverter Defibrillator Register. A 12-lead electrocardiogram was available for 2,107 participants. Associations between MAC scores and AV conduction disturbances were assessed using multivariate regression analyses. RESULTS: MAC was present in 22.4% of the study subjects. Participants with pacemakers for an AV conduction disturbance had significantly higher MAC scores (odds ratio [OR], 1.11; 95% confidence interval [CI], 1.01-1.23) than participants without a CIED, whereas participants with a CIED for other reasons did not. Prolonged QRS-interval was significantly associated with the presence of MAC (OR, 1.45; 95% CI, 1.04-2.04), whereas prolonged PQ-interval was not. Female sex and most traditional cardiovascular risk factors were significantly associated with high MAC scores. CONCLUSIONS: MAC was associated with AV conduction disturbances, which could improve our understanding of the development of AV conduction disturbances.

Aortic valve calcification among elderly males from the general population, associated echocardiographic findings, and clinical implications.

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 < 0.0001). 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.

Cross-sectional study of aortic valve calcification and cardiovascular risk factors in older Danish men.

OBJECTIVE: Aortic 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. METHODS: Participants 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. RESULTS: 14 073 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. CONCLUSIONS: AVC 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 NUMBER: NCT03946410 and ISRCTN12157806.

Prognostic importance of left atrial size measured by non-contrast cardiac computed tomography - A DANCAVAS study.

BACKGROUND: Enlargement of left atrium (LA) is a valuable marker of cardiovascular events, and LA size is readily available while performing non-contrast cardiac computed tomography (NCCT) for preventive purposes. We aimed to evaluate the predictive value of a single LA area from NCCT in a population-based cohort. METHOD: Mainly men aged 60-75 years from DANCAVAS were included. Traditional risk factors were recorded, and an NCCT scan performed at baseline. Coronary artery calcifications (CAC) score and the largest LA area were measured. LA was indexed to body surface area and categorised into four groups. Data on incident atrial fibrillation (AF), thromboembolic events, heart failure (HF) and death were obtained from Danish national registries. RESULTS: In total, 14,557 individuals were eligible, excluding those without LA measurement (N = 232) and with heart valve replacement (N = 197). Known AF or HF were respectively excluded from follow-up. Median follow-up time was 2.1 to 3.4 years. In total, 304 developed AF, 149 had thromboembolism, 129 developed HF and 482 died. In adjusted analysis, LA enlargement was associated with AF (HR (95% CI): large 1.99 (1.46-2.71) and very large LA 3.77 (2.31-6.14)) and HF (large 2.40 (1.50-3.85) and very large LA 6.54 (4.07-10.51)). A very large LA significantly increased mortality (HR: 2.01 (1.44-2.82)), and was associated with a two-fold increased risk of thromboembolism; however, not significantly in adjusted analysis (p = 0.09). CONCLUSION: We demonstrated that determination of LA area from NCCT was an important predictor of AF, HF and death. This knowledge could inform current risk assessment beyond CAC score.

Effects of menaquinone-7 supplementation in patients with aortic valve calcification: study protocol for a randomised controlled trial.

INTRODUCTION: Aortic stenosis is a common heart valve disease, and due to the growing elderly population, the prevalence is increasing. The disease is progressive with increasing calcification of the valve cusps. A few attempts with medical preventive treatment have failed; thus, presently, the only effective treatment of aortic stenosis is surgery. This study will examine the effect of menaquinone-7 (MK-7) supplementation on progression of aortic valve calcification (AVC). We hypothesise that MK-7 supplementation will slow down the calcification process. METHODS AND ANALYSIS: In this multicenter and double-blinded, placebo-controlled study, 400 men aged 65-74 years with substantial AVC are randomised (1:1) to treatment with MK-7 (720 µg/day) supplemented by the recommended daily dose of vitamin D (25 µg/day) or placebo treatment (no active treatment) for 2 years. Exclusion criteria are treatment with vitamin K antagonist or coagulation disorders. To evaluate AVC score, a non-contrast CT scan is performed at baseline and repeated after 12 and 24 months of follow-up. Primary outcome is difference in AVC score from baseline to follow-up at 2 years. Intention-to-treat principle is used for all analyses. ETHICS AND DISSEMINATION: There are no reported adverse effects associated with the use of MK-7. The protocol is approved by the Regional Scientific Ethical Committee for Southern Denmark (S-20170059) and the Data Protection Agency (17/19010). It is conducted in accordance with the Declaration of Helsinki. Positive as well as negative findings will be reported. TRIAL REGISTRATION NUMBER: NCT03243890.

Left ventricular mural thrombus despite treatment with dabigatran and clopidogrel.

We describe a case with severe heart failure and moderate aortic stenosis. Due to previous atrial fibrillation and ischaemic heart disease, this patient was treated with both dabigatran and clopidogrel. Despite this, a large mural thrombus was found on echocardiography. The treatment was altered to warfarin, but the thrombus did not resolve during the next eight months.Guidelines for the use of anticoagulant treatment in left ventricular thrombus are needed. Previously, a few cases presenting resistance to novel oral anticoagulants have been published and cases with thrombus formation due to dabigatran have been described. Our patient showed resistance to both dabigatran and warfarin, and there was no thrombus resolution when changing the treatment to warfarin.

Measurement of left atrial volume by 2D and 3D non-contrast computed tomography compared with cardiac magnetic resonance imaging.

BACKGROUND: Cardiac magnetic resonance imaging (MRI) is considered the gold standard for assessment of left atrial (LA) volume. We assessed the feasibility of evaluating LA volume using 3D non-contrast computed tomography (NCCT). Furthermore, since manual tracing of LA volume is time consuming, we evaluated the accuracy of the LA area using 2D NCCT imaging for LA volume assessment. METHODS: MRI and NCCT imaging were performed in 69 patients before and one year after aortic valve replacement. In 3D MRI and 3D NCCT, each slice was manually traced, excluding the pulmonary veins and atrial appendage, and multiplied by slice spacing, thus generating a measure of LA volume. The LA volume was indexed to body surface area. On 2D NCCT, the largest axial cross-section LA area was traced manually. RESULTS: The mean LA volume was 102 ±â€¯28 ml in MRI compared with 103 ±â€¯28 ml in 3D NCCT. 3D NCCT showed good agreement with MRI measurements (mean difference -0.7 ml/m2; 95% confidence interval (CI) -2.2 to 0.9). By Bland-Altman, 3D NCCT also showed good agreement with MRI (limits of agreement: -18.7-17.4 ml/m2). Furthermore, good correlation was found between 2D NCCT and 3D NCCT LA volume (r = 0.93). CONCLUSION: 2D and 3D measurements of LA volume in non-contrast computed tomography are feasible and accurate.

Endothelin-1 shifts the mediator of bradykinin-induced relaxation from NO to H2 O2 in resistance arteries from patients with cardiovascular disease.

BACKGROUND AND PURPOSE: We tested the hypothesis that in resistance arteries from cardiovascular disease (CVD) patients, effects of an endothelium-dependent vasodilator depend on the contractile stimulus. EXPERIMENTAL APPROACH: Arteries dissected from parietal pericardium of cardiothoracic surgery patients were studied by myography and imaging techniques. Segments were sub-maximally contracted by K(+) , the TxA2 analogue U46619 or endothelin-1 (ET-1). KEY RESULTS: Relaxing effects of Na-nitroprusside were comparable, but those of bradykinin (BK) were bigger in the presence of ET-1 compared with K(+) or U46619. BK-induced relaxation was (i) abolished by L-NAME in K(+) -contracted arteries, (ii) partly inhibited by L-NAME in the presence of U46619 and (iii) not altered by indomethacin, L-NAME plus inhibitors of small and intermediate conductance calcium-activated K(+) channels, but attenuated by catalase, in ET-1-contracted arteries. This catalase-sensitive relaxation was unaffected by inhibitors of NADPH oxidases or allopurinol. Exogenous H2 O2 caused a larger relaxation of ET-1-induced contractions than those evoked by K(+) or U46619 in the presence of inhibitors of other endothelium-derived relaxing factors. Catalase-sensitive staining of cellular ROS with CellROX Deep Red was significantly increased in the presence of both 1 µM BK and 2 nM ET-1 but not either peptide alone. CONCLUSIONS AND IMPLICATIONS: In resistance arteries from patients with CVD, exogenous ET-1 shifts the mediator of relaxing responses to the endothelium-dependent vasodilator BK from NO to H2 O2 and neither NADPH oxidases, xanthine oxidase nor NOS appear to be involved in this effect. This might have consequences for endothelial dysfunction in conditions where intra-arterial levels of ET-1 are enhanced.