Aims Cardiovascular disease (CVD) remains the leading cause of morbidity and mortality worldwide. Current guidelines recommend algorithms to estimate 10-year CVD risk in apparently healthy individuals, but their precision remains limited. Whether the integration of imaging and genetic information can improve individual risk stratification remains uncertain.Methods and results The CVRISK-IT trial is a multicentre, open-label, randomized controlled study designed to enhance CVD prevention through precision-based risk assessment and targeted communication strategies. A total of 30 000 adults aged 40-80 years without previous CVD or diabetes are recruited across Italy. In Phase I, participants undergo a baseline assessment including clinical examination, blood sampling, and calculation of CVD risk. Approximately 12 000 eligible enter Phase II and are randomized equally into four intervention arms: (i) standard care (control), (ii) imaging-based risk refinement (coronary artery calcium score or carotid ultrasound), (iii) polygenic risk score (PRS), or (iv) combination of imaging and PRS. The co-primary endpoints are differences in estimated 10-year CVD risk at 12 months and the incidence of major CVD events, CVD mortality, and all-cause mortality at 5 years. Secondary endpoints include differences in CVD risk factors, prescription and adherence to preventive drug adherence, and behavioural or psychological measures.Conclusion By prospectively evaluating the feasibility and impact of incorporating genetic and imaging information in CVD risk stratification, CVRISK-IT will generate robust evidence to inform its integration into routine screening practices and public health policy.Trial registration ClinicalTrials.gov Identifier NCT06832644.

BACKGROUND:Circulating proprotein convertase subtilisin/kexin type 9 (PCSK9) is a crucial regulator of cholesterol metabolism. Loss-of-function variants in PCSK9 are associated with lower levels of circulating low-density lipoprotein cholesterol (LDL-C) and reduced cardiovascular disease (CVD) risk, while gain-of-function variants correlate with elevated LDL-C concentrations and increased CVD risk. This study investigated whether genetically determined LDL-C levels, proxied by four PCSK9 genetic variants, influence common carotid artery atherosclerosis. METHODS:The analysis included 3040 European participants (mean age 64.2±5.4 years; 45.8% men) at high cardiovascular risk from the IMPROVE Study, alongside 49 088 individuals of white British ancestry (mean age 55.2±7.6 years; 47.9% men) from the UK Biobank (UKB). Ultrasonographic measurements of common carotid intima-media thickness (CC-IMTmean, CC-IMTmax, CC-IMTmean-max) were obtained. Four lipid-level affecting genetic variants in the PCSK9 locus were selected for analysis, both individually and in a standardised Lipid-Lowering Allelic Score (LLAS), to assess their effects on LDL-C and PCSK9 levels in the IMPROVE cohort and on ultrasonographic measures in both IMPROVE and UKB. RESULTS:In the IMPROVE cohort, PCSK9 variants (rs11206510, rs2479409, rs11591147, rs11583680) exhibited expected effect directions, although not all statistically significant, on LDL-C and PCSK9 levels. The LLAS was negatively correlated with CC-IMTmean, CC-IMTmax and CC-IMTmean-max among women in IMPROVE, and among men and overall in UKB (all p<0.05). Effect sizes were comparable between cohorts. CONCLUSIONS:Genetic variants in the PCSK9 locus influence LDL-C levels and CC-IMT, in keeping with proven benefits of PCSK9 inhibitors on atherosclerotic cardiovascular events.

Background and aims: Circulating PCSK9 is a crucial regulator of cholesterol metabolism. Loss-of-function variants in PCSK9 are associated with lower levels of circulating low-density lipoprotein cholesterol (LDL-C) and reduced cardiovascular disease (CVD) risk, while gain-of-function variants correlate with elevated LDL-C concentrations and increased CVD risk. This study investigated whether genetically determined LDL-C levels, proxied by four PCSK9 genetic variants, influence common carotid artery atherosclerosis. Methods: The analysis included 3,040 European participants (mean age 64.2 {plus minus} 5.4 years; 45.8% men) at high cardiovascular risk from the IMPROVE study, alongside 49,088 individuals of white British ancestry (mean age 55.2 {plus minus} 7.6 years; 47.9% men) from the UK Biobank (UKB). Ultrasonographic measurements of common carotid intima-media thickness (CC-IMTmean, CC-IMTmax, CC-IMTmean-max) were obtained. Four lipid-level affecting genetic variants in the PCSK9 locus were selected for analysis, both individually and in a standardized polygenic risk score (PRS), to assess their effects on LDL-C and PCSK9 levels in the IMPROVE cohort and on ultrasonographic measures in both IMPROVE and UKB. Results: In the IMPROVE cohort, PCSK9 variants (rs11206510, rs2479409, rs11591147, rs11583680) exhibited expected effect directions, albeit not all statistically significant, on LDL-C and PCSK9 levels. The PRS was negatively correlated with CC-IMTmean, CC-IMTmax, and CC-IMTmean-max among women in IMPROVE, and among men and overall in UKB (all P < 0.05). Effect sizes were comparable between cohorts. Conclusions: Genetic variants in the PCSK9 locus influence LDL-C levels and CC-IMT, in keeping with proven benefits of PCSK9 inhibitors on atherosclerotic cardiovascular events. ### Competing Interest Statement A.Gallo has received honoraria related to consulting, research, and or speaker activities from Amarin, Amryt, Amgen, Astrazeneca, Eli-Lilly, MSD, Novo-Nordisk, Novartis, Sanofi/Regeneron, Servier, Viatris and Ultragenyx. P. Welsh declares grant support from Astrazeneca, Roche diagnostics, Boehringer Ingelheim, and Novartis outside the submitted work and honoraria from Novo Nordisk and Raisio outside the submitted work. N. Sattar reports consulting/speaker honoraria from Abbott Laboratories, AbbVie, Amgen, AstraZeneca, Boehringer Ingelheim, Eli Lilly, Hanmi Pharmaceuticals, Janssen, Menarini-Ricerche, Novartis, Novo Nordisk, Pfizer, Roche Diagnostics, and Sanofi; and institutional research support from AstraZeneca, Boehringer Ingelheim, Novartis, and Roche Diagnostics D. Coggi, J. Ward, B. Gigante, M. Amato, D.M. Lyall, B. Frigerio, A. Ravani, D. Sansaro, F. Veglia, N. Capra, K. Savonen, M. Pirro, D.J. Mulder, N. Ferri, M.G. Lupo, C. Macchi, M. Ruscica, R. Baetta, E. Tremoli, D. Baldassarre and R.J. Strawbridge declare no conflict of interest. ### Funding Statement At least in part, this study uses data derived from a project funded by the European Commission, Fifth Framework Programme (Contract number: QLG1-CT-2002-00896; to E. Tremoli, D. Baldassarre) and by the Ministry of Health, Italy (RC2018 MMP4.9 ID: 2634520; RC2019 MPP 4D ID: 2755475; to D. Baldassarre). The UKB was founded by the Wellcome Trust, Medical Research Council, Department of Health, Scottish Government and Northwest Regional Development Agency. UKB was also funded by the Welsh Assembly Government and the British Heart Foundation. Data collection was supported by UKB. R.J. Strawbridge is supported by a UKRI Innovation-HDR-UK (MR/S003061/1) and the University of Glasgow Lord Kelvin Adam Smith Fellowships. The funders were not involved in the interpretation or presentation of the findings. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The IMPROVE study complies with the rules of Good Clinical Practice and with the ethical principles established in the Declaration of Helsinki, and was approved by 6 independent ethics committees, i.e.: the Regional Ethics Review Board at Karolinska Institutet, Stockholm, Sweden (approval ID: Dnr 2003/03-115, 17 February 2003); the Institutional Review Board of the Health Department of the Hospital "Ospedale Niguarda Ca' Granda", Milan, Italy (approval ID: 2042/03, 3 June 2003); the Medical Ethics Review Committee, Academic Hospital Groningen, Groningen, the Netherlands (approval ID: METc 2003/054, 12 May 2003); the Ethics Committee of the Umbrian Health Authorities, Perugia, Italy (approval ID: N 2725/03/A, 6 February 2003); the Consultative Committee for the Protection of Persons in Biomedical Research (CCPPRB) at Hôspital Pitie Salpétrière, Paris, France (approval ID: CCP61-03, 2003); the Research Ethics Committee of the University of Kuopio and Kuopio University Hospital, Kuopio, Finland (approval ID: 140/2002, 10 October 2002). All patients provided informed consent twice; once for general participation in the study and once for genotyping. All participants in UKB provided informed electronic consent and UKB was granted ethical approval by the North-West Multicentre Research Ethics Committee on the 29th of June 2021 (Ref 21/NW/0157). This study was conducted under project #71392, (P.I. Strawbridge). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes UKB data is available via application to UKB (https://www.ukbiobank.ac.uk/). IMPROVE is not available for sharing. Analytic code is available upon request from the corresponding author.

Background/Objectives: Carotid intima-media thickness (CIMT) has long been used as an index of subclinical atherosclerosis, but its role as a risk modifier in cardiovascular (CV) risk optimization has recently been questioned due to methodological problems, such as lack of protocol standardization and scanning difficulties. In this multicentre, longitudinal, and observational study, we tested the predictive ability of two new CIMT variables detectable with a simplified, quick, and easy-to-standardize protocol. Methods: CIMT was measured in 3165 subjects from six centers, in five European countries, belonging to the IMPROVE study. The two variables tested were the average of two maximal CIMT measures taken, from a single angle, in the right and left common carotids (1CC-IMTmean-of-2-max) or bifurcations (BIF-IMTmean-of-2-max). The ability to predict CV events, on top of the SCORE2/SCORE2-OP risk algorithm, was quantified by the time-dependent increase in the receiver operating characteristic (ROC) area under the curve (AUC). Results: During a median follow-up of 7.1 years, 367 cardio-, cerebro-, and peripheral-vascular events were registered. Both CIMT variables tested were associated with CV risk, but 1CC-IMTmean-of-2-max was also able to significantly increase the ROC AUC over the risk score (+0.017, p = 0.014). The result was stable after running several sensitivity analyses. Conclusions: 1CC-IMTmean-of-2-max is able to significantly improve the predictive capacity of SCORE2/SCORE2-OP. Being based on a simple and easily standardized measurement protocol, this new variable is a promising candidate for application in mass screening and risk assessment in primary prevention.

The process of adipogenesis involves the differentiation of preadipocytes into mature adipocytes. Excessive adipogenesis promotes obesity, a condition that increasingly threatens global health and contributes to the rapid rise of obesity-related diseases. We have recently shown that prenylcysteine oxidase 1 (PCYOX1) is a regulator of atherosclerosis-disease mechanisms, which acts through mechanisms not exclusively related to its pro-oxidant activity. To address the role of PCYOX1 in the adipogenic process, we extended our previous observations confirming that Pcyox1−/−/Apoe−/− mice fed a high-fat diet for 8 or 12 weeks showed significantly lower body weight, when compared to Pcyox1+/+/Apoe−/− mice, due to an evident reduction in visceral adipose content. We herein assessed the role of PCYOX1 in adipogenesis. Here, we found that PCYOX1 is expressed in adipose tissue, and, independently from its pro-oxidant enzymatic activity, is critical for adipogenesis. Pcyox1 gene silencing completely prevented the differentiation of 3T3-L1 preadipocytes, by acting as an upstream regulator of several key players, such as FABP4, PPARγ, C/EBPα. Proteomic analysis, performed by quantitative label-free mass spectrometry, further strengthened the role of PCYOX1 in adipogenesis by expanding the list of its downstream targets. Finally, the absence of Pcyox1 reduces the inflammatory markers in adipose tissue. These findings render PCYOX1 a novel adipogenic factor with possible pathophysiological or therapeutic potential.

Introduction Prevention of cardiovascular disease (CVD) is of key importance in reducing morbidity, disability and mortality worldwide. Observational studies suggest that digital health interventions can be an effective strategy to reduce cardiovascular (CV) risk. However, evidence from large randomised clinical trials is lacking. Methods and analysis The CV-PREVITAL study is a multicentre, prospective, randomised, controlled, open-label interventional trial designed to compare the effectiveness of an educational and motivational mobile health (mHealth) intervention versus usual care in reducing CV risk. The intervention aims at improving diet, physical activity, sleep quality, psycho-behavioural aspects, as well as promoting smoking cessation and adherence to pharmacological treatment for CV risk factors. The trial aims to enrol approximately 80 000 subjects without overt CVDs referring to general practitioners’ offices, community pharmacies or clinics of Scientific Institute for Research, Hospitalization and Health Care (Italian acronym IRCCS) affiliated with the Italian Cardiology Network. All participants are evaluated at baseline and after 12 months to assess the effectiveness of the intervention on short-term endpoints, namely improvement in CV risk score and reduction of major CV risk factors. Beyond the funded life of the study, a long-term (7 years) follow-up is also planned to assess the effectiveness of the intervention on the incidence of major adverse CV events. A series of ancillary studies designed to evaluate the effect of the mHealth intervention on additional risk biomarkers are also performed. Ethics and dissemination This study received ethics approval from the ethics committee of the coordinating centre (Monzino Cardiology Center; R1256/20-CCM 1319) and from all other relevant IRBs and ethics committees. Findings are disseminated through scientific meetings and peer-reviewed journals and via social media. Partners are informed about the study’s course and findings through regular meetings. Trial registration number NCT05339841.

The research into the pathophysiology of atherosclerosis has considerably increased our understanding of the disease complexity, but still many questions remain unanswered, both mechanistically and pharmacologically. Here, we provided evidence that the pro-oxidant enzyme Prenylcysteine Oxidase 1 (PCYOX1), in the human atherosclerotic lesions, is both synthesized locally and transported within the subintimal space by proatherogenic lipoproteins accumulating in the arterial wall during atherogenesis. Further, Pcyox1 deficiency in Apoe-/- mice retards atheroprogression, is associated with decreased features of lesion vulnerability and lower levels of lipid peroxidation, reduces plasma lipid levels and inflammation. PCYOX1 silencing in vitro affects the cellular proteome by influencing multiple functions related to inflammation, oxidative stress, and platelet adhesion. Collectively, these findings identify the pro-oxidant enzyme PCYOX1 as an emerging player in atherogenesis and, therefore, understanding the biology and mechanisms of all functions of this unique enzyme is likely to provide additional therapeutic opportunities in addressing atherosclerosis.

Background and Aims: The research into the pathophysiology of atherosclerosis has considerably increased our understanding of the complexity of the disease, but still many questions remain unanswered, and the burden of recurrent cardiovascular events remains unacceptable, despite optimal treatment with contemporary intervention and pharmacologic agents. Our previous findings that prenylcysteine oxidase 1 (PCYOX1) represents a novel lipoprotein-associated protein, and that lipoproteins can themselves generate the reactive oxygen species H2O2 through the action of PCYOX1, prompted us to investigate the biological role of this unique enzyme in the development of atherosclerosis.

Platelets are a heterogeneous small anucleate blood cell population with a central role both in physiological haemostasis and in pathological states, spanning from thrombosis to inflammation, and cancer. Recent advances in proteomic studies provided additional important information concerning the platelet biology and the response of platelets to several pathophysiological pathways. Platelets circulate systemically and can be easily isolated from human samples, making proteomic application very interesting for characterizing the complexity of platelet functions in health and disease as well as for identifying and quantifying potential platelet proteins as biomarkers and novel antiplatelet therapeutic targets. To date, the highly dynamic protein content of platelets has been studied in resting and activated platelets, and several subproteomes have been characterized including platelet-derived microparticles, platelet granules, platelet releasates, platelet membrane proteins, and specific platelet post-translational modifications. In this review, a critical overview is provided on principal platelet proteomic studies focused on platelet biology from signaling to granules content, platelet proteome changes in several diseases, and the impact of drugs on platelet functions. Moreover, recent advances in quantitative platelet proteomics are discussed, emphasizing the importance of targeted quantification methods for more precise, robust and accurate quantification of selected proteins, which might be used as biomarkers for disease diagnosis, prognosis and therapy, and their strong clinical impact in the near future.

Clinical and preclinical studies over the past 3 decades have uncovered a multitude of signaling pathways involved in the initiation and progression of atherosclerosis. From these studies, signaling by proteins of the Wnt family has recently emerged as an important player in the development of atherosclerosis. Wnt signaling is characterized by a large number of ligands, receptors, and coreceptors and can be regulated at many different levels. Among Wnt modulators, the evolutionary conserved Dkk (Dickkopf) proteins, and especially Dkk-1, the founding member of the family, are the best characterized. The role of Dkks in the pathophysiology of the arterial wall is only partially understood, but their involvement in atherosclerosis is becoming increasingly evident. This review introduces recent key findings on Dkk proteins and their functions in atherosclerosis and discusses the potential importance of modulating Dkk signaling as part of a novel, improved strategy for preventing and treating atherosclerosis-related diseases. Visual Overview— An online visual overview is available for this article.

This study shows that DKK-1, a member of the Dickkopf family and a regulator of the Wnt pathways, represents a novel target of statins which, through the inhibition of HMG-CoA reductase and of non-steroidal isoprenoid intermediates, exert extra-beneficial effect in preventing atherosclerosis beyond their effect on the lipid profile. We found that atorvastatin downregulates DKK-1 protein (−88.3 ± 4.1%) and mRNA expression (−90 ± 4.2%) through the inhibition of Cdc42, Rho and Rac geranylgeranylated proteins. Further, a combined approach based on the integration of label-free quantitative mass spectrometry based-proteomics and gene silencing allowed us to demonstrate that DKK-1 itself mediates, at least in part, statin effects on human endothelial cells. Indeed, DKK-1 is responsible for the regulation of the 21% of the statin-modulated proteins, which include, among others, clusterin/apoJ, plasminogen activator inhibitor type 1 (PAI-1), myristoylated alanine-rich C-kinase substrate (MARCKS), and pentraxin 3 (PTX3). The Gene Ontology enrichment annotation revealed that DKK-1 is also a potential mediator of the extracellular matrix organization, platelet activation and response to wounding processes induced by statin. Finally, we found that plasma level of DKK-1 from cholesterol-fed rabbits treated with atorvastatin (2.5 mg/kg/day for 8 weeks) was lower (−42 ± 23%) than that of control animals. Thus, DKK-1 is not only a target of statin but it directly regulates the expression of molecules involved in a plethora of biological functions, thus expanding its role, which has been so far restricted mainly to cancer.

Cardiovascular diseases (CVDs) represent the most important cause of mortality in women and in men. Contrary to the long-standing notion that the effects of the major risk factors on CVD outcomes are the same in both sexes, recent evidence recognizes new, potentially independent, sex/gender-related risk factors for CVDs, and sex/gender-differences in the clinical presentation of CVDs have been demonstrated. Furthermore, some therapeutic options may not be equally effective and safe in men and women. In this context, proteomics offers an extremely useful and versatile analytical platform for biomedical researches that expand from the screening of early diagnostic and prognostic biomarkers to the investigation of the molecular mechanisms underlying CDVs. In this review, we summarized the current applications of proteomics in the cardiovascular field, with emphasis on sex and gender-related differences in CVDs.SIGNIFICANCE:Increasing evidence supports the profound effect of sex and gender on cardiovascular physio-pathology and the response to drugs. A clear understanding of the mechanisms underlying sexual dimorphisms in CVDs would not only improve our knowledge of the etiology of these diseases, but could also inform health policy makers and guideline committees in tailoring specific interventions for the prevention, treatment and management of CVDs in both men and women.

Proteomic-based techniques provide a powerful tool for identifying the full spectrum of protein targets of a drug, elucidating its mechanism(s) of action, and identifying biomarkers of its efficacy and safety. Herein, we outline the technological advancements in the field, and illustrate the contribution of proteomics to the definition of the pharmacological profile of statins, which represent the cornerstone of the prevention and treatment of cardiovascular diseases (CVDs). Statins act by inhibiting 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase, thus reducing cholesterol biosynthesis and consequently enhancing the clearance of low-density lipoproteins from the blood; however, HMG-CoA reductase inhibition can result in a multitude of additional effects beyond lipid lowering, known as 'pleiotropic effects'. The case of statins highlights the unique contribution of proteomics to the target profiling of a drug molecule.

Background and aims: Proprotein convertase subtilisin kexin type 9 (PCSK9) induces degradation of the low-density lipoprotein-receptor (LDLR). Smooth muscle cells (SMCs) in human atherosclerotic plaques and cultured SMCs express PCSK9. The present study aimed at defining the role of PCSK9 on vascular response to injury.Methods: Carotid neointimal lesions were induced by positioning a non-occlusive collar in PCSK9 knockout (PCSK9(-/-)) and wild type littermate (PCSK9(+/+)) mice.Results: In PCSK9(-/-) mice, we observed a significantly less intimal thickening (p < 0.05), a lower intimal media ratio (p < 0.02), and a tendency to higher lumen area, compared to PCSK9(+/+) mice. When compared with PCSK9(-/-), lesions of PCSK9(+/+) mice had a higher content of SMCs (p < 0.05) and collagen (p < 0.05), while no difference was observed in the accumulation of macrophages. PCSK9 was detectable in both left and right carotids artery in regions occupied by medial and neointimal SMCs. SMCs freshly isolated from PCSK9(-/-), when compared to PCSK9(+/+) cells, showed higher levels of alpha-smooth muscle actin (alpha-SMA; 2.24 +/- 0.36 fold; p < 0.01) and myosin heavy chain II (MHC-II; 8.65 +/- 1.55 fold; p < 0.01), and lower levels of caldesmon mRNA (-54 +/- 14%; p < 0.01). PCSK9(-/-) cells also showed a slower proliferation rate, and an impaired migratory capacity and G1/S progression of the cell cycle. The reconstitution of PCSK9 expression, by retroviral infection of PCSK9(-/-) SMCs, led to a downregulation of a-SMA (-56 +/- 2%; p < 0.01), MHC-II(-45% +/- 25.5 fold: p = 0.06) and calponin (-25% +/- 0.8 fold: p < 0.05) and induction of caldesmon mRNA (1.46 +/- 0.3 fold; p < 0.05). Proliferation rate of SMCs PCSK9(-/-) was significantly lower compared to PCSK9 reconstituted cells.Conclusions: Taken together, the present results suggest that PCSK9, by sustaining SMC synthetic phenotype, proliferation, and migration, may play a pro-atherogenic role in the arterial wall. (C) 2016 Elsevier Ireland Ltd. All rights reserved.