Although a previous study has suggested that a genetic variant in the LPA region was associated with the presence of aortic valve stenosis (AVS), no prospective study has suggested a role for ...lipoprotein(a) levels in the pathophysiology of AVS. Our objective was to determine whether lipoprotein(a) levels and a common genetic variant that is strongly associated with lipoprotein(a) levels are associated with an increased risk of developing AVS.
Serum lipoprotein(a) levels were measured in 17 553 participants of the European Prospective Investigation into Cancer (EPIC)-Norfolk study. Among these study participants, 118 developed AVS during a mean follow-up of 11.7 years. The rs10455872 genetic variant in LPA was genotyped in 14 735 study participants, who simultaneously had lipoprotein(a) level measurements, and in a replication study of 379 patients with echocardiography-confirmed AVS and 404 controls. In EPIC-Norfolk, compared with participants in the bottom lipoprotein(a) tertile, those in the top lipoprotein(a) tertile had a higher risk of AVS (hazard ratio, 1.57; 95% confidence interval, 1.02-2.42) after adjusting for age, sex, and smoking. Compared with rs10455872 AA homozygotes, carriers of 1 or 2 G alleles were at increased risk of AVS (hazard ratio, 1.78; 95% confidence interval, 1.11-2.87, versus hazard ratio, 4.83; 95% confidence interval, 1.77-13.20, respectively). In the replication study, the genetic variant rs10455872 also showed a positive association with AVS (odds ratio, 1.57; 95% confidence interval, 1.10-2.26).
Patients with high lipoprotein(a) levels are at increased risk for AVS. The rs10455872 variant, which is associated with higher lipoprotein(a) levels, is also associated with increased risk of AVS, suggesting that this association may be causal.
OBJECTIVE—The mechanisms underlying the pathogenesis of aortic valve calcification remain unclear. With accumulating evidence demonstrating that valve calcification recapitulates bone development, ...the crucial roles of noncanonical Wnt ligands WNT5a, WNT5b, and WNT11 in osteogenesis make them critical targets in the study of aortic valve calcification.
APPROACH AND RESULTS—Using immunohistochemistry, real-time qPCR, Western blotting, and tissue culture, we examined the tissue distribution of WNT5a, WNT5b, and WNT11 in noncalcified and calcified aortic valves and their effects on human aortic valve interstitial cells (HAVICs). Only focal strong immunostaining for WNT5a was seen in and around areas of calcification. Abundant immunostaining for WNT5b and WNT11 was seen in inflammatory cells, fibrosis, and activated myofibroblasts in areas of calcified foci. There was significant correlation between WNT5b and WNT11 overall staining and presence of calcification, lipid score, fibrosis, and microvessels (P<0.05). Real-time qPCR and Western blotting revealed abundant expression of both Wnts in stenotic aortic valves, particularly in bicuspid valves. Incubation of HAVICs from noncalcified valves with the 3 noncanonical Wnts significantly increased cell apoptosis and calcification (P<0.05). Treatment of HAVICs with the mitogen-activated protein kinase-38β and GSK3β inhibitors significantly reduced their mineralization (P<0.01). Raman spectroscopy identified the inorganic phosphate deposits as hydroxyapatite and showed a significant increase in hydroxyapatite deposition in HAVICs in response to WNT5a and WNT11 (P<0.05). Similar crystallinity was seen in the deposits found in HAVICs treated with Wnts and in calcified human aortic valves.
CONCLUSIONS—These findings suggest a potential role for noncanonical Wnt signaling in the pathogenesis of aortic valve calcification.
The acute respiratory distress syndrome (ARDS) is characterized by intense dysregulated inflammation leading to acute lung injury (ALI) and respiratory failure. There are no effective pharmacologic ...therapies for ARDS. Colchicine is a low-cost, widely available drug, effective in the treatment of inflammatory conditions. We studied the effects of colchicine pre-treatment on oleic acid-induced ARDS in rats. Rats were treated with colchicine (1 mg/kg) or placebo for three days prior to intravenous oleic acid-induced ALI (150 mg/kg). Four hours later they were studied and compared to a sham group. Colchicine reduced the area of histological lung injury by 61%, reduced lung edema, and markedly improved oxygenation by increasing PaO2/FiO2 from 66 ± 13 mmHg (mean ± SEM) to 246 ± 45 mmHg compared to 380 ± 18 mmHg in sham animals. Colchicine also reduced PaCO2 and respiratory acidosis. Lung neutrophil recruitment, assessed by myeloperoxidase immunostaining, was greatly increased after injury from 1.16 ± 0.19% to 8.86 ± 0.66% and significantly reduced by colchicine to 5.95 ± 1.13%. Increased lung NETosis was also reduced by therapy. Circulating leukocytosis after ALI was not reduced by colchicine therapy, but neutrophils reactivity and CD4 and CD8 cell surface expression on lymphocyte populations were restored. Colchicine reduces ALI and respiratory failure in experimental ARDS in relation with reduced lung neutrophil recruitment and reduced circulating leukocyte activation. This study supports the clinical development of colchicine for the prevention of ARDS in conditions causing ALI.
The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) 3CL protease is a promising target for inhibition of viral replication by interaction with a cysteine residue (Cys145) at its ...catalytic site. Dalcetrapib exerts its lipid-modulating effect by binding covalently to cysteine 13 of a cholesteryl ester transfer protein. Because 12 free cysteine residues are present in the 3CL protease, we investigated the potential of dalcetrapib to inhibit 3CL protease activity and SARS-CoV-2 replication. Molecular docking investigations suggested that dalcetrapib-thiol binds to the catalytic site of the 3CL protease with a delta G value of −8.5 kcal/mol. Dalcetrapib inhibited both 3CL protease activity in vitro and viral replication in Vero E6 cells with IC50 values of 14.4 ± 3.3 μM and an EC50 of 17.5 ± 3.5 μM (mean ± SD). Near-complete inhibition of protease activity persisted despite 1000-fold dilution after ultrafiltration with a nominal dalcetrapib-thiol concentration of approximately 100 times below the IC50 of 14.4 μM, suggesting stable protease–drug interaction. The inhibitory effect of dalcetrapib on the SARS-CoV-2 3CL protease and viral replication warrants its clinical evaluation for the treatment of COVID-19.
The large HDL particles generated by administration of cholesteryl ester transfer protein inhibitors (CETPi) remain poorly characterized, despite their potential importance in the routing of ...cholesterol to the liver for excretion, which is the last step of the reverse cholesterol transport. Thus, the effects of the CETPi dalcetrapib and anacetrapib on HDL particle composition were studied in rabbits and humans. The association of rabbit HDL to the LDL receptor (LDLr) in vitro was also evaluated. New Zealand White rabbits receiving atorvastatin were treated with dalcetrapib or anacetrapib. A subset of patients from the dal-PLAQUE-2 study treated with dalcetrapib or placebo were also studied. In rabbits, dalcetrapib and anacetrapib increased HDL-C by more than 58% (P < 0.01) and in turn raised large apo E-containing HDL by 66% (P < 0.001) and 59% (P < 0.01), respectively. Additionally, HDL from CETPi-treated rabbits competed with human LDL for binding to the LDLr on HepG2 cells more than control HDL (P < 0.01). In humans, dalcetrapib increased concentrations of large HDL particles (+69%, P < 0.001) and apo B-depleted plasma apo E (+24%, P < 0.001), leading to the formation of apo E-containing HDL (+47%, P < 0.001) devoid of apo A-I. Overall, in rabbits and humans, CETPi increased large apo E-containing HDL particle concentration, which can interact with hepatic LDLr. The catabolism of these particles may depend on an adequate level of LDLr to contribute to reverse cholesterol transport.
Left ventricular diastolic dysfunction (LVDD) is present in more than 50% of patients suffering from heart failure. LVDD animal models are limited and its underlying mechanisms remain largely ...unknown. Aortic valve stenosis (AVS) may cause LVDD, and we recently reported LVDD in an AVS rabbit model. Here we aimed to develop a rabbit model of LVDD without AVS.
Rabbits were fed with a 0.5% cholesterol-enriched diet (n = 9) or normal diet (n = 8) until they developed LVDD defined by a value of the echocardiographic parameter E/Em ratio higher than the mean at baseline + 2SD. Rabbits were then fed a 0.2% cholesterol-enriched diet for 4 weeks (average total diet duration: 20 weeks). Detailed cardiac structure and function measurements were assessed by echocardiography at baseline, weeks 8, 12 and 14 to 20, when applicable. Histological analyses and RT-qPCR were performed on LV samples.
The hypercholesterolemic diet induced LVDD without systolic dysfunction or AVS, as shown by multiple echocardiographic parameters, including early filling mitral peak velocity and deceleration rate, Em/Am ratio and E/Em ratio (all p<0.05), and by increased cardiac mRNA expression of brain natriuretic peptide (Bnp). Cardiac expression of mRNA for Nox2, Vcam1, Mmp12, Mmp12/Timp1, Il1b and Col1/Col3 ratios was also higher in these rabbits (p<0.05). In contrast, cardiac Sod2 mRNA expression was reduced in hypercholesterolemic rabbits compared to controls.
Rabbits fed with a cholesterol-enriched diet develop LVDD with preserved systolic function and evidence of cardiac inflammation and oxidative stress. This rabbit model may be used in future studies to test treatment strategies against LVDD.
Inhibition of cholesteryl ester transfer protein (CETP) increases HDL cholesterol (HDL-C) levels. However, the circulating CETP level varies and the impact of its inhibition in species with high CETP ...levels on HDL structure and function remains poorly characterized. This study investigated the effects of dalcetrapib and anacetrapib, the two CETP inhibitors (CETPis) currently being tested in large clinical outcome trials, on HDL particle subclass distribution and cholesterol efflux capacity of serum in rabbits and monkeys. New Zealand White rabbits and vervet monkeys received dalcetrapib and anacetrapib. In rabbits, CETPis increased HDL-C, raised small and large α-migrating HDL, and increased ABCA1-induced cholesterol efflux. In vervet monkeys, although anacetrapib produced similar results, dalcetrapib caused opposite effects because the LDL-C level was increased by 42% and HDL-C decreased by 48% (P < 0.01). The levels of α- and preβ-HDL were reduced by 16% (P < 0.001) and 69% (P < 0.01), resulting in a decrease of the serum cholesterol efflux capacity. CETPis modulate the plasma levels of mature and small HDL in vivo and consequently the cholesterol efflux capacity. The opposite effects of dalcetrapib in different species indicate that its impact on HDL metabolism could vary greatly according to the metabolic environment.
The progression of coronary atherosclerosis is an active and regulated process. The Wnt signaling pathway is thought to play an active role in the pathogenesis of several cardiovascular diseases; ...however, a better understanding of this system in atherosclerosis is yet to be unraveled.
In this study, real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting were used to quantify the expression of Wnt3a, Wnt5a, and Wnt5b in the human coronary plaque, and immunohistochemistry was used to identify sites of local expression. To determine the pathologic significance of increased Wnt, human vascular smooth muscle cells (vSMCs) were treated with Wnt3a, Wnt5a, and Wnt5b recombinant proteins and assessed for changes in cell differentiation and function.
RT-PCR and Western blotting showed a significant increase in the expression of Wnt3a, Wnt5a, Wnt5b, and their receptors in diseased coronary arteries compared with that in non-diseased coronary arteries. Immunohistochemistry revealed an abundant expression of Wnt3a and Wnt5b in diseased coronary arteries, which contrasted with little or no signals in normal coronary arteries. Immunostaining of Wnt3a and Wnt5b was found largely in inflammatory cells and myointimal cells. The treatment of vSMCs with Wnt3a, Wnt5a, and Wnt5b resulted in increased vSMC differentiation, migration, calcification, oxidative stress, and impaired cholesterol handling.
This study demonstrates the upregulation of three important members of canonical and non-canonical Wnt signaling pathways and their receptors in coronary atherosclerosis and shows an important role for these molecules in plaque development through increased cellular remodeling and impaired cholesterol handling.
OBJECTIVE—Studies have shown that high-density lipoprotein (HDL)–raising compounds induce regression of aortic valve stenosis (AVS) in animal models. However, whether patients with AVS have an ...impaired HDL metabolism is unknown.
APPROACH AND RESULTS—A total of 1435 single nucleotide polymorphisms in genes associated with HDL cholesterol levels (in or around GALNT2, LPL, ABCA1, APOA5, SCARB1, LIPC, CETP, LCAT, LIPG, APOC4, and PLTP) were genotyped in 382 patients with echocardiography-confirmed AVS (aortic jet velocity ≥2.5 m/s) and 401 controls. After control for multiple testing, none of the genetic variants showed a positive association with case/control status (adjusted P≥0.05 for all single nucleotide polymorphisms tested). In a subsample of this cohort, HDL cholesterol levels, apolipoprotein AI levels, lecithin-cholesterol acyltransferase activity, pre–β-HDL, HDL size, and 4 parameters of cholesterol efflux capacity were measured in apolipoprotein B–depleted serum samples from 86 patients with and 86 patients without AVS. Cholesterol efflux capacity was measured using J774 macrophages with and without stimulation of ATP-binding cassette A-1 expression by cAMP, and HepG2 hepatocytes for scavenger receptor class B type 1–mediated efflux. None of these parameters were different between cases and controls. However, compared with patients without coronary artery disease, sera from patients with coronary artery disease had lower HDL cholesterol levels, scavenger receptor class B type 1–mediated efflux, and HDL size (P≤0.003), independently of the presence or absence of AVS.
CONCLUSIONS—Results of the present study suggest that, based on HDL genetics and HDL functionality, HDL metabolism does not seem to predict the risk of AVS. Because of our limited sample size, additional studies are needed to confirm these findings.
Polymorphisms in the adenylate cyclase 9 (ADCY9) gene influence the benefits of the cholesteryl ester transfer protein (CETP) modulator dalcetrapib on cardiovascular events after acute coronary ...syndrome. We hypothesized that Adcy9 inactivation could improve cardiac function and remodelling following myocardial infarction (MI) in absence of CETP activity.
Wild-type (WT) and Adcy9-inactivated (Adcy9Gt/Gt) male mice, transgenic or not for human CETP (tgCETP+/–), were subjected to MI by permanent left anterior descending coronary artery ligation and studied for 4 weeks. Left ventricular (LV) function was assessed by echocardiography at baseline, 1, and 4 weeks after MI. At sacrifice, blood, spleen and bone marrow cells were collected for flow cytometry analysis, and hearts were harvested for histologic analyses.
All mice developed LV hypertrophy, dilation, and systolic dysfunction, but Adcy9Gt/Gt mice exhibited reduced pathologic LV remodelling and better LV function compared with WT mice. There were no differences between tgCETP+/– and Adcy9Gt/Gt tgCETP+/– mice, which both exhibited intermediate responses. Histologic analyses showed smaller cardiomyocyte size, reduced infarct size, and preserved myocardial capillary density in the infarct border zone in Adcy9Gt/Gt vs WT mice. Count of bone marrow T cells and B cells were significantly increased in Adcy9Gt/Gt mice compared with the other genotypes.
Adcy9 inactivation reduced infarct size, pathologic remodelling, and cardiac dysfunction. These changes were accompanied by preserved myocardial capillary density and increased adaptive immune response. Most of the benefits of Adcy9 inactivation were only observed in the absence of CETP.
Les polymorphismes du gène de l’adénylate cyclase 9 (ADCY9) influent sur les bienfaits du traitement par le dalcétrapib, un modulateur de la protéine de transfert des esters de cholestérol (CETP), sur les événements cardiovasculaires après un syndrome coronarien aigu. Nous avons émis l’hypothèse que l’inactivation du gène ADCY9 pourrait améliorer le fonctionnement et le remodelage cardiaques après un infarctus du myocarde (IM) en l’absence d’activité de la CETP.
Un IM a été provoqué par ligature de l’artère coronaire descendante antérieure gauche chez des souris mâles de type sauvage (WT) et porteuses du gène ADCY9 inactivé (ADCY9Gt/Gt), transgéniques ou non pour le gène de la CETP humaine (tgCETP+/–), et ces souris ont été étudiées pendant 4 semaines. Le fonctionnement du ventricule gauche (VG) a été évalué par échocardiographie au départ, ainsi qu’à 1 et 4 semaines après l’IM. Au moment du sacrifice, la rate, du sang et des cellules de moelle osseuse ont été prélevés à des fins d’analyses par cytométrie de flux, ainsi que le cœur à des fins d’analyses histologiques.
Toutes les souris présentaient une hypertrophie, une dilatation et un dysfonctionnement systolique du VG, mais les souris ADCY9Gt/Gt présentaient un remodelage pathologique moins important et un meilleur fonctionnement du VG comparativement aux souris de type sauvage. Aucune différence n’a été observée entre les souris tgCETP+/– et ADCY9Gt/Gt tgCETP+/–, qui présentaient des réponses intermédiaires. Les analyses histologiques ont révélé une taille plus petite des cardiomyocytes, une taille d’infarctus inférieure, et une densité capillaire myocardique préservée en bordure de l’infarctus chez les souris ADCY9Gt/Gt comparativement aux souris de type sauvage. Les nombres de lymphocytes T et B dans la moelle étaient sensiblement accrus chez les souris ADCY9Gt/Gt comparativement aux autres génotypes.
L’inactivation du gène ADCY9 a réduit la taille de l’infarctus, le remodelage pathologique et le dysfonctionnement cardiaque. Ces changements étaient associés à une densité capillaire myocardique préservée et à une réponse immunitaire adaptative accrue. La plupart des avantages de l’inactivation du gène ADCY9 n’ont été observés qu’en l’absence d’activité de la CETP.
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