Low Density Lipoproteins Promote Unstable Calcium Handling Accompanied by Reduced SERCA2 and Connexin-40 Expression in Cardiomyocytes

The damaging effects of high plasma levels of cholesterol in the cardiovascular system are widely known, but little attention has been paid to direct effects on cardiomyocyte function. We therefore aimed at testing the hypothesis that Low Density Lipoprotein (LDL) cholesterol affects calcium dynamics and signal propagation in cultured atrial myocytes. For this purpose, mRNA and protein expression levels were determined by real time PCR and western blot analysis, respectively, and intracellular calcium was visualized in fluo-4 loaded atrial HL-1 myocyte cultures subjected to field stimulation. At low stimulation frequencies all cultures had uniform calcium transients at all tested LDL concentrations. However, 500 μg LDL/mL maximally reduced the calcium transient amplitude by 43% from 0.30±0.04 to 0.17±0.02 (p<0.05). Moreover, LDL-cholesterol dose-dependently increased the fraction of alternating and irregular beat-to-beat responses observed when the stimulation interval was shortened. This effect was linked to a concurrent reduction in SERCA2, RyR2, IP3RI and IP3RII mRNA levels. SERCA2 protein levels were also reduced by 43% at 200 μg LDL/mL (p<0.05) and SR calcium loading was reduced by 38±6% (p<0.001). By contrast, HDL-cholesterol had no significant effect on SERCA expression or SR calcium loading. LDL-cholesterol also slowed the conduction velocity of the calcium signal from 3.2+0.2 mm/s without LDL to 1.7±0.1 mm/s with 500 μg LDL/mL (p<0.05). This coincided with a reduction in Cx40 expression (by 44±3%; p<0.05 for mRNA and by 79±2%; p<0.05 for Cx40 protein at 200 μg/ml LDL) whereas the Cx-43 expression did not significantly change. In conclusion, LDL-cholesterol destabilizes calcium handling in cultured atrial myocytes subjected to rapid pacing by reducing SERCA2 and Cx40 expression and by slowing the conduction velocity of the calcium signal. © 2013 Barriga et al.This work was supported by grants from the Spanish Ministry of Science and Innovation: SAF2011-30312 and CNIC2009-08 to Leif Hove Madsen, DPI2009-06999 to Rau´ l Benı´tez and from the Spanish Ministry of Health and Consume: FIS PI11/00747 to Vicenta Llorente. Funding from Redes de Investigacio´n Cooperativa, Instituto de Salud Carlos III, red REDINSCOR RD06-0003-0000 and RD06-0003-0015 is also acknowledged. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer Reviewe

Monomeric C-reactive protein - A key molecule driving development of Alzheimer's disease associated with brain ischaemia?

M. Slevin et al.Alzheimer's disease (AD) increases dramatically in patients with ischaemic stroke. Monomeric C-reactive protein (mCRP) appears in the ECM of ischaemic tissue after stroke, associating with microvasculature, neurons and AD-plaques, Aβ, also, being able to dissociate native-CRP into inflammatory, mCRP in vivo. Here, mCRP injected into the hippocampal region of mice was retained within the retrosplenial tract of the dorsal 3rd ventrical and surrounding major vessels. Mice developed behavioural/cognitive deficits within 1 month, concomitant with mCRP staining within abnormal looking neurons expressing p-tau and in beta-amyloid 1-42-plaque positive regions. mCRP co-localised with CD105 in microvessels suggesting angiogenesis. Phospho-arrays/Western blotting identified signalling activation in endothelial cells and neurons through p-IRS-1, p-Tau and p-ERK1/2-which was blocked following pre-incubation with mCRP-antibody. mCRP increased vascular monolayer permeability and gap junctions, increased NCAM expression and produced haemorrhagic angiogenesis in mouse matrigel implants. mCRP induced tau244-372 aggregation and assembly in vitro. IHC study of human AD/stroke patients revealed co-localization of mCRP with Aβ plaques, tau-like fibrils and IRS-1/P-Tau positive neurons and high mCRP-levels spreading from infarcted core regions matched reduced expression of Aβ/Tau. mCRP may be responsible for promoting dementia after ischaemia and mCRP clearance could inform therapeutic avenues to reduce the risk of future dementia.We would like to thank the Fundacion BBVA for their generous support of Professor Mark Slevin through the award of BBVA Chair in Clinical Biomedicine at the ICCC, St Pau Hospital, Barcelona. This work was also supported by the Spanish Ministry of Science (SAF2009-01237 to J.K.), Spanish Ministry Project-CSD2010-00045 to C.S. and RyC (RyC2007-01466 to M.B.-P.) and the Almajmaah University Stroke ChairPeer Reviewe

Functional And Cognitive Decline Is Associated With Increased Endothelial Cell Inflammation And Platelet Activation. Liquid Biopsy Of Microvesicles In Community-Dwelling Octogenarians.

Increased life expectancy is usually associated with comorbidities, such as cardio andcerebrovascular disease causing impaired functionality. A common underlying cause ofthese comorbidities is vascular inflammation and injury. Elevated levels of circulatingmicrovesicles (cMV), as a product of a hemostatic and inflammatory cell activation,could be direct mapping of an imbalanced hemostasis. In this manuscript, we aimedto investigate by liquid biopsy whether successful aging can be discriminated bycMV levels and phenotype. To this purpose, we included 135 community-dwellingoctogenarians in a cross-sectional study. Successful aging was defined as goodfunctional (Barthel Index>90 points, and Lawton index score>7/4 points forwomen and men, respectively) and cognitive status (Spanish version of the Mini-Mental State Examination -MEC->24 points) and no need for institutionalization.Total, annexin V positive (AV+), and AV−cMV from different cell origins from thevascular compartment were phenotypically characterized and quantified from fastingplasma samples by flow cytometry. Successful aging was associated with lowerplasma concentrations of total and AV+CD141+/CD41+-CD61+, and PAC1+/AV+,CD141+/AV+, and CD36+/AV−cMV. From these phenotypes, ROC curve analysesrevealed that CD141+/AV+and CD141+/CD41+-CD61+/AV+endothelial- and platelet-derived cMV discriminate successful and non-successful aging with an AUC (95%CI) of0.655 (0.551, 0.758),P= 0.005, and 0.638 (0.535, 0.741),P= 0.013, respectively. In conclusion, successful aging is associated with low levels of cMV released by endothelialcells and platelets, indicating lower endothelial cell inflammation and platelet activation.Our results contribute to the understanding of the link between unsuccessful aging,cognitive decline and vascular cell inflammatory disturbances

A novel truncated form of apolipoprotein A-I transported by dense LDL is increased in diabetic patients

Diabetic (DM) patients have exacerbated atherosclerosis and high CVD burden. Changes in lipid metabolism, lipoprotein structure, and dysfunctional HDL are characteristics of diabetes. Our aim was to investigate whether serum ApoA-I, the main protein in HDL, was biochemically modified in DM patients. By using proteomic technologies, we have identified a 26 kDa ApoA-I form in serum. MS analysis revealed this 26 kDa form as a novel truncated variant lacking amino acids 1-38, ApoA-IΔ(1-38). DM patients show a 2-fold increase in ApoA-IΔ(1-38) over nondiabetic individuals. ApoA-IΔ(1-38) is found in LDL, but not in VLDL or HDL, with an increase in LDL3 and LDL4 subfractions. To identify candidate mechanisms of ApoA-I truncation, we investigated potentially involved enzymes by in silico data mining, and tested the most probable molecule in an established animal model of diabetes. We have found increased hepatic cathepsin D activity as one of the potential proteases involved in ApoA-I truncation. Cathepsin D-cleaved ApoA-I exhibited increased LDL binding affinity and decreased antioxidant activity against LDL oxidation. In conclusion, we show for the first time: a) presence of a novel truncated ApoA-I form, ApoA-IΔ(1-38), in human serum; b) ApoA-IΔ(1-38) is transported by LDL; c) ApoA-IΔ(1-38) is increased in dense LDL fractions of DM patients; and d) cathepsin D-ApoA-I truncation may lead to ApoA-IΔ(1-38) binding to LDLs, increasing their susceptibility to oxidation and contributing to the high cardiovascular risk of DM patients. This research was originally published in Lipid Research. A novel truncated form of apolipoprotein A-I transported by dense LDL is increased in diabetic patients. Journal of Lipid Research. 2015. 56: 1762-1773 © the American Society for Biochemistry and Molecular Biology.Peer Reviewe

Tratamiento antiarrítmico actual de la fibrilación auricular no valvular en España: datos del Registro FANTASIIA

[Abstract] Introduction and objectives. Recently, there have been many developments in the management of nonvalvular atrial fibrillation, antiarrhythmic and anticoagulant therapy, and nonpharmacological treatment, but these developments are not applied immediately in clinical practice. The aim of this study was to identify the overall management and antiarrhythmic therapy used in the current general population of patients with nonvalvular atrial fibrillation in Spain. Methods. A prospective, observational study of 1318 consecutive anticoagulated patients with nonvalvular atrial fibrillation, recruited between June 2013 and March 2014. We analyzed the patients’ general characteristics, management, and antiarrhythmic therapy. Results. Mean age was 73.8 ± 9.4 years; 42.5% were women. Atrial fibrillation was paroxysmal in 28% of the patients, permanent in 50%, persistent in 17.6%, long-standing persistent in 4.5%, and new-onset in 66 patients (5%). A rhythm control strategy was chosen in 39.4% of the patients and rate control in 60.6%. Beta-blockers were prescribed in 60.2% of the patients, digoxin in 19.5%, and calcium channel antagonists in 10.7%. The antiarrhythmic agents used were amiodarone (12.6%), flecainide (8.9%), propafenone (0.4%), sotalol (0.5%), and dronedarone (2.3%). Cardioversion had been performed previously in 41.9% of the patients, ablation in 3.4%, and atrial appendage closure in 0.2%. Conclusions. Currently, patients with nonvalvular atrial fibrillation in Spain are managed mainly with rate control, and beta-blockers in particular. They receive few antiarrhythmic agents and only a very small number of these patients undergo nonpharmacological treatments.[Resumen] Introducción y objetivos. Recientemente se han producido numerosas novedades en el manejo de la fibrilación auricular no valvular y el tratamiento antiarrítmico, anticoagulante y no farmacológico empleado, pero su aplicación a la clínica no es inmediata. El objetivo del trabajo es conocer las características generales de manejo y tratamiento antiarrítmico de una población general de pacientes con fibrilación auricular no valvular actualmente en España. Métodos. Estudio observacional y prospectivo de 1.318 pacientes consecutivos con fibrilación auricular no valvular, anticoagulados y reclutados entre junio de 2013 y marzo de 2014. Se analizan sus características generales, el manejo y el tratamiento antiarrítmico utilizado. Resultados. La media de edad era 73,8 ± 9,4 años; eran mujeres el 42,5%. La fibrilación auricular fue paroxística en el 28% de los casos, permanente en el 50%, persistente en el 17,6%, persistente de larga duración en el 4,5% y de novo en 66 pacientes (5%). Se eligió control del ritmo en el 39,4% de los casos y de frecuencia en el 60,6%. Tomaron bloqueadores beta el 60,2%, digoxina el 19,5% y antagonistas del calcio el 10,7%. Los antiarrítmicos empleados fueron amiodarona (12,6%), flecainida (8,9%), propafenona (0,4%), sotalol (0,5%) y dronedarona (2,3%). Se realizó cardioversión previa en el 41,9%, ablación en el 3,4% y cierre de orejuela en el 0,2%. Conclusiones. Actualmente en nuestro país se maneja a los pacientes con fibrilación auricular no valvular preferentemente con control de frecuencia, sobre todo con bloqueadores beta, reciben pocos antiarrítmicos y se los somete en muy baja proporción a tratamientos no farmacológicos

Anticoagulantes orales directos frente a antagonistas de la vitamina K en pacientes del «mundo real» con fibrilación auricular no valvular: estudio FANTASIIA

Observational study[Abstract] Introduction and objectives: To compare the long-term results of direct oral anticoagulants (DOAC) vs vitamin K antagonists (VKA) in real-world-patients with nonvalvular atrial fibrillation (NVAF) in a nationwide, prospective study. Methods: The FANTASIIA registry prospectively included outpatients with AF anticoagulated with DOAC or VKA (per protocol, proportion of VKA and DOAC 4:1), consecutively recruited from June 2013 to October 2014 in Spain. The incidence of major events was analyzed and compared according to the anticoagulant treatment received. Results: A total of 2178 patients were included in the study (mean age 73.8±9.4 years), and 43.8% were women. Of these, 533 (24.5%) received DOAC and 1645 (75.5%) VKA. After a median follow up of 32.4 months, patients receiving DOAC vs those receiving VKA had lower rates of stroke-0.40 (95%CI, 0.17-0.97) vs 1.07 (95%CI,0.79-1.46) patients/y, P=.032-, severe bleedings-2.13 (95%CI, 1.45-3.13) vs 3.28 (95%CI, 2.75-3.93) patients/y; P = .044-, cardiovascular death-1.20 (95%CI, 0.72-1.99) vs 2.45 (95%CI, 2.00-3.00) patients/y; P = .009-, and all-cause death-3.77 (95%CI, 2.83-5.01) vs 5.54 (95%CI, 4.83-6.34) patients/y; P = .016-. In a modified Cox regression model by the Andersen-Gill method for multiple events, hazard ratios for patients receiving DOAC were: 0.42 (0.16-1.07) for stroke; 0.47 (0.20-1.16) for total embolisms; 0.76 (0.50-1.15) for severe bleedings; 0.67 (0.39-1.18) for cardiovascular death; 0.86 (0.62-1.19) for all-cause death, and 0.82 (0.64-1.05) for the combined event consisting of stroke, embolism, severe bleeding, and all-cause death. Conclusions: Compared with VKA, DOAC is associated with a trend to a lower incidence of all major events, including death, in patients with NVAF in Spain.[Resumen] Introducción y objetivos. Comparar los resultados a largo plazo de los anticoagulantes orales directos (ACOD) frente a los antagonistas de la vitamina K (AVK) en pacientes del mundo real con fibrilación auricular no valvular (FANV) en un estudio nacional prospectivo. Métodos. El estudio FANTASIIA incluyó consecutivamente a pacientes ambulatorios con FANV anticoagulados con ACOD o AVK desde junio de 2013 hasta octubre de 2014. Se compararon las tasas de eventos según el anticoagulante administrado. Resultados. Se incluyó a 2.178 pacientes (edad, 73,8 ± 9,4 años; el 43,8% mujeres); de ellos, 533 (24,5%) recibían ACOD y 1.645 (75,5%), AVK. Tras una mediana de seguimiento de 32,4 meses, los pacientes con ACOD tuvieron tasas más bajas de ictus —0,40 (IC95%, 0,17-0,97) frente a 1,07 (IC95%, 0,79-1,46) pacientes/año; p = 0,032—, hemorragias mayores —2,13 (IC95%, 1,45-3,13) frente a 3,28 (IC95%, 2,75-3,93) pacientes/año; p = 0,044—, muerte cardiovascular —1,20 (IC95%, 0,72-1,99) frente a 2,45 (IC95%, 2,00-3,00) pacientes/año; p = 0,009— y muerte total —3,77 (IC95%, 2,83-5,01) frente a 5,54 (IC95%, 4,83-6,34) pacientes/año; p = 0,016—. En el análisis de Cox modificado según el método de Andersen-Gill para datos con múltiples eventos, las razones de riesgos instantáneos para los pacientes con ACOD fueron 0,42 (0,16-1,07) para el ictus; 0,47 (0,20-1,16) para la embolia sistémica en general; 0,76 (0,50-1,15) para las hemorragias mayores; 0,67 (0,39-1,18) para la muerte cardiovascular; 0,86 (0,62-1,19) para la mortalidad total y 0,82 (0,64-1,05) para el combinado de ictus, embolias, hemorragias mayores y muerte. Conclusiones. El tratamiento con ACOD se asocia con una tendencia a una menor tasa de todos los eventos graves, incluida la mortalidad, en relación con los AVK en pacientes con FANV en España

Herramientas bioinformáticas para el análisis de resultados de experimentos de expresión génica

En este estudio se propone una metodología que combina datos de expresión diferencial con información de expresión entre proteínas. El objetivo es enriquecer los resultados de un experimento de expresión con nuevos genes que no fueron tenidos en cuenta en el diseño del experimento pero que podrían tener cierta relevancia en los procesos que muestran expresión diferencial. El método se basa en el estudio de las vías proteómicas que conectan genes con expresión diferencial significativa.Peer ReviewedPostprint (author’s final draft

Imaging of early inflammation in low-to-moderate carotid stenosis by 18-FDG-PET

It is not clear if 18FDG-PET can be useful for detection of inflammation in low to moderate carotid stenosis. We studied 15 patients scheduled for endarterectomy with contralateral carotids with less than 50% stenosis. 18-FDG-PET was performed prior to CEA and 3 months following surgery. FDG-uptake values were calculated based on maximum standardized uptake value (SUV) and corresponding uptake ratios. We confirmed by CD68 macrophage staining that FDG accumulation corresponds to active inflammation (R=0.8 p less than 0.005). We found significant correlation between the FDG-uptake in the carotids scheduled for CEA and contralateral carotids with low to moderate stenosis (R=0.9 p less than 0.001). The FDG uptake ratio in the contralateral arteries remained stable on the follow-up imaging (1.15+/-0.2 vs. 1.14+/-0.1, R=0.7 p=0.006). We did not find correlation between FDG uptake and symptomatic or asymptomatic patients, degree of carotid stenosis and vascular risk factors. This is a prospective, preliminary in vivo study demonstrating that low to moderate carotid atherosclerosis can be detected using 18-FDG-PET imaging and highlights the truly systemic nature of atherosclerosis

Peripheral artery disease and clinical outcomes in patients with atrial fibrillation: A report from the FANTASIIA registry

Observational study[Abstract] Background: Atrial fibrillation (AF) and peripheral artery disease (PAD) are common conditions that increase cardiovascular risk. We determined the association between PAD and prognosis in a cohort of real-world patients receiving oral anticoagulant therapy for nonvalvular AF. Methods: We prospectively included 1956 patients (mean age 73.8 ± 9.5 years, 44.0% women) receiving oral anticoagulant therapy for AF. Clinical characteristics were collected at baseline. Patients were followed for a period of 3 years. Survival analysis and multivariable regression analyses were performed to assess variables related to death, stroke, bleeding, myocardial infarction and major adverse cardiovascular events (MACE). Results: Patients with PAD (n = 118; 6%) exhibited higher rates of cardiovascular risk factors and cardiovascular diseases. After 3 years of follow-up, there were a total of 255 deaths (no PAD 233, vs PAD 22), 45 strokes (43 vs 2), 146 major bleedings (136 vs 10) and 168 MACE (148 vs 20). On univariate analysis, there was a higher risk of cardiovascular mortality (2.02%/year no PAD vs 4.08%/year PAD, P = .02), myocardial infarction (0.99%/year no PAD vs 2.43%/year PAD, P = .02) and MACE (3.18%/year no PAD vs 6.99%/year PAD, P < .01). There was no statistically significant association with these events after multivariable adjustment. Conclusions: In a large cohort of anticoagulated patients with AF, the presence of PAD represents a higher risk subgroup and is associated with worse crude outcomes. The exact contribution of the PAD independently of other cardiovascular diseases or risk factors requires further investigation.Instituto de Salud Carlos III; RD16/11/00420Instituto de Salud Carlos III; RD12/0042/0068Instituto de Salud Carlos III; RD12/0042/0010Instituto de Salud Carlos III; RD12/0042/0069Instituto de Salud Carlos III; RD12/0042/006

Predictive value of the SAMe-TTR2R2 to predict quality of oral anticoagulation in atrial fibrillation: a prospective validation in the multicentre spanish observational registry FANTASIIA

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