Unravelling the immunomodulatory role of apple phenolic rich extracts on human THP-1- derived macrophages using multiplatform metabolomics

Financiado para publicación en acceso aberto: Universidade de Vigo/CISUGApples represent a significant source of dietary phenolic compounds with evidenced anti-inflammatory and immunomodulatory activities. Nevertheless, the effect of the whole apple matrix on human macrophages is unknown. In this context, our study attempts to evaluate the effect of apple-derived phenolic compounds-rich extracts (pulp, peel and leaf) on IL-1β production in THP-1-differentiated macrophages and derived metabolic alterations through untargeted metabolomics. Our results have showed that apple pulp treatment inhibited the release of the pro-inflammatory cytokine IL-1β induced by LPS in THP-1 macrophages by ELISA analysis. Metabolomics demonstrate that different proportions of phenolic compounds led to differential alterations in the metabolism of THP-1 macrophages. Indeed, apple extracts promoted alterations in lipid, carbohydrate, amino acid and vitamins as well as cofactors metabolism. Specifically, leaf extracts were characterized by alteration of galactose metabolism while the extracts derived from the fruit showed predominant alterations in lipids metabolism. All extracts mimicked the response observed under normal conditions in LPS-stimulated macrophages, inhibiting LPS response. Thus, the phenolic enriched extracts from apples will be a good source of natural compounds with a beneficial effect against inflammation, and they may be applied as a food supplement and/or functional ingredient for the treatment of inflammatory diseasesFondo Europeo de Desarrollo Regional (FEDER) | Ref. 0377_IBERPHENOL_6_EFundação para a Ciência e a Tecnologia | Ref. UIDB/50006/2020Fundação para a Ciência e a Tecnologia | Ref. PTDC/SAU-NUT/30448/2017Fundação para a Ciência e a Tecnologia | Ref. SFRH/BPD/86173/201

Metoprolol exerts a non-class effect against ischaemia-reperfusion injury by abrogating exacerbated inflammation.

Clinical guidelines recommend early intravenous β-blockers during ongoing myocardial infarction; however, it is unknown whether all β-blockers exert a similar cardioprotective effect. We experimentally compared three clinically approved intravenous β-blockers. Mice undergoing 45 min/24 h ischaemia-reperfusion (I/R) received vehicle, metoprolol, atenolol, or propranolol at min 35. The effect on neutrophil infiltration was tested in three models of exacerbated inflammation. Neutrophil migration was evaluated in vitro and in vivo by intravital microscopy. The effect of β-blockers on the conformation of the β1 adrenergic receptor was studied in silico. Of the tested β-blockers, only metoprolol ameliorated I/R injury [infarct size (IS) = 18.0% ± 0.03% for metoprolol vs. 35.9% ± 0.03% for vehicle; P < 0.01]. Atenolol and propranolol had no effect on IS. In the three exacerbated inflammation models, neutrophil infiltration was significantly attenuated only in the presence of metoprolol (60%, 50%, and 70% reductions vs. vehicle in myocardial I/R injury, thioglycolate-induced peritonitis, and lipopolysaccharide-induced acute lung injury, respectively). Migration studies confirmed the particular ability of metoprolol to disrupt neutrophil dynamics. In silico analysis indicated different intracellular β1 adrenergic receptor conformational changes when bound to metoprolol than to the other two β-blockers. Metoprolol exerts a disruptive action on neutrophil dynamics during exacerbated inflammation, resulting in an infarct-limiting effect not observed with atenolol or propranolol. The differential effect of β-blockers may be related to distinct conformational changes in the β1 adrenergic receptor upon metoprolol binding. If these data are confirmed in a clinical trial, metoprolol should become the intravenous β-blocker of choice for patients with ongoing infarction.Ministry of Science and Innovation (‘RETOS 2019’ grant N_ PID2019-107332RB-I00), Instituto de Salud Carlos III (ISCIII; PI16/02110), and European Regional Development Fund (# AC16/00021), Comunidad de Madrid (S2017/BMD-3867 RENIM-CM). B.I. is supported by an ERCCoG grant (819775). E.O. is supported by funds from the Comunidad de Madrid Programa de Atraccion de Talento (2017-T1/BMD-5185). A.C-M. and R.V-G are supported by fellowships from the Ministerio de Ciencia e Innovacion (MCN) and ISCIII (FPU2017/01932 and PFIS FI17/00045). D.V.L. is supported by an Iniciativa de Empleo Juvenil grant (PEJ-2017-TL/BMD-6463) from the Comunidad de Madrid. The CNIC is supported by the ISCIII, the MCN, and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (SEV-2015-0505).S

The extracellular proteins of Lactobacillus acidophilus DSM 20079T display anti-inflammatory effect in both in piglets, healthy human donors and Crohn’s Disease patients

Lactobacillus genus includes both probiotic and representative strains of the human gut microbiota. Independent studies have reported on the anti-inflammatory properties of different Lactobacillus strains, although we are far from understanding the underlying molecular interplay. In this work we show that a daily administration of Lactobacillus acidophilus DSM20079T (DSM20079) to healthy piglets resulted in plasmatic increases of the anti-inflammatory IL10, whilst IL12 and the pro-inflammatory ratio IL12+TNFα/IL10 decreased. The extracellular protein fraction of DSM20079 was identified as the responsible for the crosstalk interaction that elicited these tolerogenic effects. This strain was able to activate innate immune pathways in dendritic cells and to decrease the production of pro-inflammatory cytokines in both CD4+/CD8+ T cell subsets in healthy donors and in Crohn’s Disease patients. The tolerogenic effect exerted by the extracellular proteins of this strain suggests their potential use as coadjutant for therapeutic applications targeting chronic inflammatory illnesses.Asociación Española Contra el Cancer | Ref. PS-2016Fundação para a Ciência e a Tecnologia | | Ref. UID/BIO/04469/2013Agencia Estatal de Investigación | Ref. AGL2016-78311-RPrincipado de Asturias | Ref. PCTI 2018–2020Xunta de Galicia | Ref. ED431C2018/5

Non-Invasive Assessment of Pulmonary Vasculopathy

Right heart catheterization remains necessary for the diagnosis of pulmonary hypertension and, therefore, for the prognostic evaluation of patients with chronic heart failure. The non-invaSive Assessment of Pulmonary vasculoPathy in Heart failure (SAPPHIRE) study was designed to assess the feasibility and prognostic relevance of a non-invasive evaluation of the pulmonary artery vasculature in patients with heart failure and pulmonary hypertension. Patients will undergo a right heart catheterization, cardiac resonance imaging, and a pulmonary function test in order to identify structural and functional parameters allowing the identification of combined pre- and postcapillary pulmonary hypertension, and correlate these findings with the hemodynamic dataThis research was funded by European Regional Development Fund and the Carlos III Research Institute through a grant of the Health Strategy Action (PI17/01569).S

In silico and functional analyses of immunomodulatory peptides encrypted in the human gut metaproteome

This work supports the massive presence of potential immunomodulatory peptides in the human gut metaproteome. These peptides were identified through the MAHMI database as potentially anti-inflammatory, and sixteen of them synthesized for characterize their mechanism of action. From them, peptide HM14 was encrypted in an extracellular protein produced by Bifidobacterium longum, a common member of the human microbiota, and displayed the highest anti-inflammatory capability. Molecular mechanism of action of HM14 pointed to a specific interaction between this immunomodulatory peptide and antigen presenting cells, which resulted in a higher formation of iTreg cells. Moreover, HM14 was effective in decreasing pro-inflammatory parameters in PBMCs isolated from a cohort of Crohn's patients. Finally, non-targeted metabolomics confirmed the ability of HM14 to modulate the metabolic activity of PBMCs to fulfil its energy and biosynthetic requirements. Overall, our combined in silico/multiomics approach supports the human gut metaproteome as a source for immunomodulatory peptides.Ministerio de Economía y Competitividad | Ref. AGL2013-44761-PAgencia Estatal de Investigación | Ref. AGL2016-78311-

Determinants of Progression and Regression of Subclinical Atherosclerosis Over 6 Years.

BACKGROUND Atherosclerosis is a systemic disease that frequently begins early in life. However, knowledge about the temporal disease dynamics (ie, progression or regression) of human subclinical atherosclerosis and their determinants is scarce. OBJECTIVES This study sought to investigate early subclinical atherosclerosis disease dynamics within a cohort of middle-aged, asymptomatic individuals by using multiterritorial 3-dimensional vascular ultrasound (3DVUS) imaging. METHODS A total of 3,471 participants from the PESA (Progression of Early Subclinical Atherosclerosis) cohort study (baseline age 40-55 years; 36% female) underwent 3 serial 3DVUS imaging assessments of peripheral arteries at 3-year intervals. Subclinical atherosclerosis was quantified as global plaque volume (mm3) (bilateral carotid and femoral plaque burden). Multivariable logistic regression models for progression and regression were developed using stepwise forward variable selection. RESULTS Baseline to 6-year subclinical atherosclerosis progression occurred in 32.7% of the cohort (17.5% presenting with incident disease and 15.2% progressing from prevalent disease at enrollment). Regression was observed in 8.0% of those patients with baseline disease. The effects of higher low-density lipoprotein cholesterol (LDL-C) and elevated systolic blood pressure (SBP) on 6-year subclinical atherosclerosis progression risk were more pronounced among participants in the youngest age stratum (Pinteraction = 0.04 and 0.02, respectively). CONCLUSIONS Over 6 years, subclinical atherosclerosis progressed in one-third of middle-age asymptomatic subjects. Atherosclerosis regression is possible in early stages of the disease. The impact of LDL-C and SBP on subclinical atherosclerosis progression was more pronounced in younger participants, a finding suggesting that the prevention of atherosclerosis and its progression could be enhanced by tighter risk factor control at younger ages, with a likely long-term impact on reducing the risk of clinical events. (Progression of Early Subclinical Atherosclerosis [PESA; also PESA-CNIC-Santander]; NCT01410318).S

Dynamic Edematous Response of the Human Heart to Myocardial Infarction Implications for Assessing Myocardial Area at Risk and Salvage

BACKGROUND: Clinical protocols aimed to characterize the post-myocardial infarction (MI) heart by cardiac magnetic resonance (CMR) need to be standardized to take account of dynamic biological phenomena evolving early after the index ischemic event. Here, we evaluated the time course of edema reaction in patients with ST-segment-elevation MI by CMR and assessed its implications for myocardium-at-risk (MaR) quantification both in patients and in a large-animal model. METHODS: A total of 16 patients with anterior ST-segment-elevation MI successfully treated by primary angioplasty and 16 matched controls were prospectively recruited. In total, 94 clinical CMR examinations were performed: patients with ST-segment-elevation MI were serially scanned (within the first 3 hours after reperfusion and at 1, 4, 7, and 40 days), and controls were scanned only once. T2 relaxation time in the myocardium (T2 mapping) and the extent of edema on T2-weighted short-tau triple inversion-recovery (ie, CMR-MaR) were evaluated at all time points. In the experimental study, 20 pigs underwent 40-minute ischemia/reperfusion followed by serial CMR examinations at 120 minutes and 1, 4, and 7 days after reperfusion. Reference MaR was assessed by contrast-multidetector computed tomography during the index coronary occlusion. Generalized linear mixed models were used to take account of repeated measurements. RESULTS: In humans, T2 relaxation time in the ischemic myocardium declines significantly from early after reperfusion to 24 hours, and then increases up to day 4, reaching a plateau from which it decreases from day 7. Consequently, edema extent measured by T2-weighted short-tau triple inversion-recovery (CMR-MaR) varied with the timing of the CMR examination. These findings were confirmed in the experimental model by showing that only CMR-MaR values for day 4 and day 7 postreperfusion, coinciding with the deferred edema wave, were similar to values measured by reference contrast-multidetector computed tomography. CONCLUSIONS: Post-MI edema in patients follows a bimodal pattern that affects CMR estimates of MaR. Dynamic changes in post-ST-segment-elevation MI edema highlight the need for standardization of CMR timing to retrospectively delineate MaR and quantify myocardial salvage. According to the present clinical and experimental data, a time window between days 4 and 7 post-MI seems a good compromise solution for standardization. Further studies are needed to study the effect of other factors on these variables.This study was partially supported by a competitive grant from the Spanish Society of Cardiology (Proyectos de Investigacion Traslacional en Cardiologia de la Sociedad Espanola de Cardiologia 2015, for the project Caracterizacion tiSUlar miocaRdica con resonancia magnetica en pacientes tras inFarto agudo de mioCardio con elevacioN de ST sometidos a angloplastia Coronaria primaria. Estudio SURF-CNIC), by a competitive grant from the Carlos III Institute of Health-Fondo de Investigacion Sanitaria- and the European Regional Development Fund (ERDF/FEDER) (PI10/02268 and PI13/01979), the Spanish Ministry of economy, industry, and competitiveness (MEIC) and ERDF/FEDER SAF2013-49663-EXP. Dr Fernandez-Jimenez holds a FICNIC fellowship from the Fundacio Jesus Serra, the Fundacion Interhospitalaria de Investigacion Cardiovascular, and the Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), and Dr Aguero is a FP7-PEOPLE-2013-ITN-Cardionext fellow. This study forms part of a Master Research Agreement between the CNIC and Philips Healthcare, and is part of a bilateral research program between Hospital de Salamanca Cardiology Department and the CNIC. This research program is part of an institutional agreement between FIIS-Fundacion Jimenez Diaz and CNIC. The CNIC is supported by the MEIC and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (MEIC award SEV-2015-0505).S

Subclinical Atherosclerosis Burden by 3D Ultrasound in Mid-Life: The PESA Study

BACKGROUND: Detection of subclinical atherosclerosis improves risk prediction beyond cardiovascular risk factors (CVRFs) and risk scores, but quantification of plaque burden may improve it further. Novel 3-dimensional vascular ultrasound (3DVUS) provides accurate volumetric quantification of plaque burden. OBJECTIVES: The authors evaluated associations between 3DVUS-based plaque burden and CVRFs and explored potential added value over simple plaque detection. METHODS: The authors included 3,860 (92.2%) PESA (Progression of Early Subclinical Atherosclerosis) study participants (age 45.8 ± 4.3 years; 63% men). Bilateral carotid and femoral territories were explored by 3DVUS to determine the number of plaques and territories affected, and to quantify global plaque burden defined as the sum of all plaque volumes. Linear regression and proportional odds models were used to evaluate associations of plaque burden with CVRFs and estimated 10-year cardiovascular risk. RESULTS: Plaque burden was higher in men (63.4 mm3 [interquartile range (IQR): 23.8 to 144.8 mm3] vs. 25.7 mm3 [IQR: 11.5 to 61.6 mm3] in women; p < 0.001), in the femoral territory (64 mm3 [IQR: 27.6 to 140.5 mm3] vs. 23.1 mm3 [IQR: 9.9 to 48.7 mm3] in the carotid territory; p < 0.001), and with increasing age (p < 0.001). Age, sex, smoking, and dyslipidemia were more strongly associated with femoral than with carotid disease burden, whereas hypertension and diabetes showed no territorial differences. Plaque burden was directly associated with estimated cardiovascular risk independently of the number of plaques or territories affected (p < 0.01). CONCLUSIONS: 3DVUS quantifies higher plaque burden in men, in the femoral territory, and with increasing age during midlife. Plaque burden correlates strongly with CVRFs, especially at the femoral level, and reflects estimated cardiovascular risk more closely than plaque detection alone. (Progression of Early Subclinical Atherosclerosis [PESA] Study; NCT01410318).The PESA study is cofunded equally by the Fundacion Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Madrid, Spain, and Banco Santander, Madrid, Spain. The study also receives funding from the Institute of Health Carlos III (PI15/02019) and the European Regional Development Fund (ERDF). The CNIC is supported by the Ministry of Economy, Industry and Competitiveness (MINECO) and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (MINECO award SEV-2015-0505). Dr. Sanchez-Gonzalez is an employee of Philips Healthcare. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose. Stephen J. Nicholls, MD, served as Guest Editor for this paperS

Longitudinal interplay between subclinical atherosclerosis, cardiovascular risk factors, and cerebral glucose metabolism in midlife: results from the PESA prospective cohort study.

BACKGROUND Cardiovascular disease and dementia often coexist at advanced stages. Yet, longitudinal studies examining the interplay between atherosclerosis and its risk factors on brain health in midlife are scarce. We aimed to characterise the longitudinal associations between cerebral glucose metabolism, subclinical atherosclerosis, and cardiovascular risk factors in middle-aged asymptomatic individuals. METHODS The Progression of Early Subclinical Atherosclerosis (PESA) study is a Spanish longitudinal observational cohort study of 4184 asymptomatic individuals aged 40-54 years (NCT01410318). Participants with subclinical atherosclerosis underwent longitudinal cerebral [18F]fluorodeoxyglucose ([18F]FDG)-PET, and annual percentage change in [18F]FDG uptake was assessed (primary outcome). Cardiovascular risk was quantified with SCORE2 and subclinical atherosclerosis with three-dimensional vascular ultrasound (exposures). Multivariate regression and linear mixed effects models were used to assess associations between outcomes and exposures. Additionally, blood-based biomarkers of neuropathology were quantified and mediation analyses were performed. Secondary analyses were corrected for multiple comparisons using the false discovery rate (FDR) approach. FINDINGS This longitudinal study included a PESA subcohort of 370 participants (median age at baseline 49·8 years [IQR 46·1-52·2]; 309 [84%] men, 61 [16%] women; median follow-up 4·7 years [IQR 4·2-5·2]). Baseline scans took place between March 6, 2013, and Jan 21, 2015, and follow-up scans between Nov 24, 2017, and Aug 7, 2019. Persistent high risk of cardiovascular disease was associated with an accelerated decline of cortical [18F]FDG uptake compared with low risk (β=-0·008 [95% CI -0·013 to -0·002]; pFDR=0·040), with plasma neurofilament light chain, a marker of neurodegeneration, mediating this association by 20% (β=0·198 [0·008 to 0·740]; pFDR=0·050). Moreover, progression of subclinical carotid atherosclerosis was associated with an additional decline in [18F]FDG uptake in Alzheimer's disease brain regions, not explained by cardiovascular risk (β=-0·269 [95% CI -0·509 to -0·027]; p=0·029). INTERPRETATION Middle-aged asymptomatic individuals with persistent high risk of cardiovascular disease and subclinical carotid atherosclerosis already present brain metabolic decline, suggesting that maintenance of cardiovascular health during midlife could contribute to reductions in neurodegenerative disease burden later in life. FUNDING Spanish Ministry of Science and Innovation, Instituto de Salud Carlos III, Santander Bank, Pro-CNIC Foundation, BrightFocus Foundation, BBVA Foundation, "la Caixa" Foundation.Spanish Ministry of Science and Innovation, Instituto de Salud Carlos III, Santander Bank, Pro-CNIC Foundation, BrightFocus Foundation, BBVA Foundation, “la Caixa” Foundation. We thank the PESA participants and the imaging, administrative, and medical PESA teams. The PESA study is equally co-funded by the Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC) and Santander Bank (Madrid, Spain) and also receives funding from the Instituto de Salud Carlos III (ISCIII), Madrid, Spain (PI15/02019), the European Regional Development Fund (ERDF—A Way to Build Europe), and the European Social Fund (ESF—Investing in Your Future). CNIC is a Severo Ochoa Center of Excellence (CEX2020- 001041-S) and is supported by the ISCIII, the Spanish Ministry for Science and Innovation, and the Pro-CNIC Foundation. CT-P was supported by a “la Caixa” Foundation fellowship (ID 100010434, LCF/BQ/DI19/11730052). MC-C was supported by a Miguel Servet type II research contract (ISCIII, CPII21/00007) and the Fondo de Investigación Sanitaria (ISCIII, PI20/00819). We acknowledge the Sephardic Foundation on Aging and other donors of the Alzheimer’s Disease Research (grant number A2022034S), a programme of the BrightFocus Foundation, for support of this research. This work was also partially produced with the support of a 2021 Leonardo Grant for Researchers and Cultural Creators from the BBVA Foundation awarded to MC-C (the Foundation takes no responsibility for the opinions, statements, and contents of this project, which are entirely the responsibility of its authors). BI was supported by the European Research Council (ERC-2018-CoG 819775-MATRIX). MS is supported by the Knut and Alice Wallenberg Foundation (Wallenberg Centre for Molecular and Translational Medicine; KAW2014.0363), the Swedish Research Council (2017-02869, 2021-02678, 2021-06545), the Swedish state under the agreement between the Swedish Government and the County Councils, the ALF-agreement (ALFGBG-813971, ALFGBG-965326), the Swedish Brain Foundation (FO2021-0311), and the Swedish Alzheimer Foundation (AF-740191). MS-C receives funding from the European Research Council (grant agreement number 948677), project “PI19/00155”, funded by ISCIII and co-funded by the EU, and a fellowship from “la Caixa” Foundation (ID 100010434) and from the EU’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement number 847648 (LCF/BQ/PR21/11840004). HZ is a Wallenberg Scholar supported by grants from the Swedish Research Council (#2022-01018), the EU’s Horizon Europe research and innovation programme under grant agreement number 101053962, Swedish State Support for Clinical Research (#ALFGBG-71320), the Alzheimer Drug Discovery Foundation, USA (#201809-2016862), the AD Strategic Fund and the Alzheimer’s Association (#ADSF-21-831376-C, #ADSF-21-831381-C, #ADSF-21-831377-C), the Bluefield Project, the Olav Thon Foundation, the Erling-Persson Family Foundation, Stiftelsen för Gamla Tjänarinnor, Hjärnfonden, Sweden (#FO2022-0270), the EU’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement number 860197 (MIRIADE), the EU Joint Programme – Neurodegenerative Disease Research (JPND2021-00694), and the UK Dementia Research Institute at UCL (UKDRI-1003). KB is supported by the Swedish Research Council (#2017-00915, #2022-00732), the Swedish state under the agreement between the Swedish Government and the County Councils, the ALFagreement (#ALFGBG-715986, #ALFGBG-965240), the Swedish Alzheimer Foundation (#AF-930351, #AF-939721, #AF-968270), Hjärnfonden, Sweden (#FO2017-0243, #ALZ2022-0006), the Alzheimer’s Association 2021 Zenith Award (ZEN-21-848495), and the Alzheimer’s Association 2022–2025 grant (SG-23-1038904 QC).S

Longitudinal interplay between subclinical atherosclerosis, cardiovascular risk factors, and cerebral glucose metabolism in midlife: results from the PESA prospective cohort study

BACKGROUND: Cardiovascular disease and dementia often coexist at advanced stages. Yet, longitudinal studies examining the interplay between atherosclerosis and its risk factors on brain health in midlife are scarce. We aimed to characterise the longitudinal associations between cerebral glucose metabolism, subclinical atherosclerosis, and cardiovascular risk factors in middle-aged asymptomatic individuals. METHODS: The Progression of Early Subclinical Atherosclerosis (PESA) study is a Spanish longitudinal observational cohort study of 4184 asymptomatic individuals aged 40-54 years (NCT01410318). Participants with subclinical atherosclerosis underwent longitudinal cerebral [18F]fluorodeoxyglucose ([18F]FDG)-PET, and annual percentage change in [18F]FDG uptake was assessed (primary outcome). Cardiovascular risk was quantified with SCORE2 and subclinical atherosclerosis with three-dimensional vascular ultrasound (exposures). Multivariate regression and linear mixed effects models were used to assess associations between outcomes and exposures. Additionally, blood-based biomarkers of neuropathology were quantified and mediation analyses were performed. Secondary analyses were corrected for multiple comparisons using the false discovery rate (FDR) approach. FINDINGS: This longitudinal study included a PESA subcohort of 370 participants (median age at baseline 49·8 years [IQR 46·1-52·2]; 309 [84%] men, 61 [16%] women; median follow-up 4·7 years [IQR 4·2-5·2]). Baseline scans took place between March 6, 2013, and Jan 21, 2015, and follow-up scans between Nov 24, 2017, and Aug 7, 2019. Persistent high risk of cardiovascular disease was associated with an accelerated decline of cortical [18F]FDG uptake compared with low risk (β=-0·008 [95% CI -0·013 to -0·002]; pFDR=0·040), with plasma neurofilament light chain, a marker of neurodegeneration, mediating this association by 20% (β=0·198 [0·008 to 0·740]; pFDR=0·050). Moreover, progression of subclinical carotid atherosclerosis was associated with an additional decline in [18F]FDG uptake in Alzheimer's disease brain regions, not explained by cardiovascular risk (β=-0·269 [95% CI -0·509 to -0·027]; p=0·029). INTERPRETATION: Middle-aged asymptomatic individuals with persistent high risk of cardiovascular disease and subclinical carotid atherosclerosis already present brain metabolic decline, suggesting that maintenance of cardiovascular health during midlife could contribute to reductions in neurodegenerative disease burden later in life. FUNDING: Spanish Ministry of Science and Innovation, Instituto de Salud Carlos III, Santander Bank, Pro-CNIC Foundation, BrightFocus Foundation, BBVA Foundation, "la Caixa" Foundation