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Aims We assessed the impact of the proprotein convertase subtilisin–kexin type 9 (PCSK9) inhibitor evolocumab on acute arterial events across all vascular territories, including coronary, cerebrovascular, and peripheral vascular beds, in patients with established atherosclerotic cardiovascular disease (ASCVD). Methods and results In the FOURIER trial, 27 564 patients with stable ASCVD on statin therapy were randomly assigned to evolocumab or placebo. Acute arterial events were a composite of acute coronary (coronary heart disease death, myocardial infarction, or urgent coronary revascularization), cerebrovascular (ischaemic stroke, transient ischaemic attack, or urgent cerebral revascularization), or peripheral vascular (acute limb ischaemia, major amputation, or urgent peripheral revascularization) events. Of the 2210 first acute arterial events, 74% were coronary, 22% were cerebrovascular, and 4% were peripheral vascular. Evolocumab reduced first acute arterial events by 19% (hazard ratio [HR] 0.81 [95% confidence interval 0.74–0.88]; P Conclusion The addition of the PCSK9 inhibitor evolocumab to statin therapy reduced acute arterial events across all vascular territories with a robust effect over time, indicating a pan-vascular impact of aggressive lipid-lowering therapy on these acute and clinically meaningful events. Clinical Trial Registration URL: https://www.clinicaltrials.gov. Unique identifier: NCT01764633.

Aims PCSK9 is secreted into the circulation, mainly by the liver, and interacts with low-density lipoprotein receptor (LDLR) homologous and non-homologous receptors, including CD36, thus favouring their intracellular degradation. As PCSK9 deficiency increases the expression of lipids and lipoprotein receptors, thus contributing to cellular lipid accumulation, we investigated whether this could affect heart metabolism and function. Methods and results Wild-type (WT), Pcsk9 KO, Liver conditional Pcsk9 KO and Pcsk9/Ldlr double KO male mice were fed for 20 weeks with a standard fat diet and then exercise resistance, muscle strength, and heart characteristics were evaluated. Pcsk9 KO presented reduced running resistance coupled to echocardiographic abnormalities suggestive of heart failure with preserved ejection fraction (HFpEF). Heart mitochondrial activity, following maximal coupled and uncoupled respiration, was reduced in Pcsk9 KO mice compared to WT mice and was coupled to major changes in cardiac metabolism together with increased expression of LDLR and CD36 and with lipid accumulation. A similar phenotype was observed in Pcsk9/Ldlr DKO, thus excluding a contribution for LDLR to cardiac impairment observed in Pcsk9 KO mice. Heart function profiling of the liver selective Pcsk9 KO model further excluded the involvement of circulating PCSK9 in the development of HFpEF, pointing to a possible role locally produced PCSK9. Concordantly, carriers of the R46L loss-of-function variant for PCSK9 presented increased left ventricular mass but similar ejection fraction compared to matched control subjects. Conclusion PCSK9 deficiency impacts cardiac lipid metabolism in an LDLR independent manner and contributes to the development of HFpEF., Graphical Abstract Impact of Pcsk9 deficiency on cardiac function and mitochondrial metabolism. Pcsk9 deficiency is associated with increased heart left ventricular thickness and reduced running performance independently of skeletal muscle alterations. Electron microscopy analysis showed increased cardiac accumulation of lipid droplets associated with a reduced density of mitochondrial cristae; this proffunctionally translated into impaired oxidative phosphorylation and mitochondrial metabolism in PCSK9 KO hearts.

Aims Statins reduce cardiovascular risk in patients with acute coronary syndrome (ACS) and normal-to-moderately impaired renal function. It is not known whether proprotein convertase subtilisin-kexin type 9 (PCSK9) inhibitors provide similar benefit across a range of renal function. We determined whether effects of the PCSK9 inhibitor alirocumab to reduce cardiovascular events and death after ACS are influenced by renal function. ................................................................................................................................................................................................... Methods ODYSSEY OUTCOMES compared alirocumab with placebo in patients with recent ACS and dyslipidaemia despite and results intensive statin treatment. Estimated glomerular filtration rate (eGFR) _90 mL/min/1.73 m (n = 7470; hazard ratio 0.784, 95% confidence interval 0.670–0.919; P = 0.003) and 60 to 60 mL/min/1.73 m 2 2 2 2, The trial was funded by Sanofi and Regeneron Pharmaceuticals, Inc.

Nucleic acid-based therapeutics are currently developed at large scale for prevention and management of cardiovascular diseases (CVDs), since: (i) genetic studies have highlighted novel therapeutic targets suggested to be causal for CVD; (ii) there is a substantial recent progress in delivery, efficacy, and safety of nucleic acid-based therapies; (iii) they enable effective modulation of therapeutic targets that cannot be sufficiently or optimally addressed using traditional small molecule drugs or antibodies. Nucleic acid-based therapeutics include (i) RNA-targeted therapeutics for gene silencing; (ii) microRNA-modulating and epigenetic therapies; (iii) gene therapies; and (iv) genome-editing approaches (e.g. CRISPR-Cas-based): (i) RNA-targeted therapeutics: several large-scale clinical development programmes, using antisense oligonucleotides (ASO) or short interfering RNA (siRNA) therapeutics for prevention and management of CVD have been initiated. These include ASO and/or siRNA molecules to lower apolipoprotein (a) [apo(a)], proprotein convertase subtilisin/kexin type 9 (PCSK9), apoCIII, ANGPTL3, or transthyretin (TTR) for prevention and treatment of patients with atherosclerotic CVD or TTR amyloidosis. (ii) MicroRNA-modulating and epigenetic therapies: novel potential therapeutic targets are continually arising from human non-coding genome and epigenetic research. First microRNA-based therapeutics or therapies targeting epigenetic regulatory pathways are in clinical studies. (iii) Gene therapies: EMA/FDA have approved gene therapies for non-cardiac monogenic diseases and LDL receptor gene therapy is currently being examined in patients with homozygous hypercholesterolaemia. In experimental studies, gene therapy has significantly improved cardiac function in heart failure animal models. (iv) Genome editing approaches: these technologies, such as using CRISPR-Cas, have proven powerful in stem cells, however, important challenges are remaining, e.g. low rates of homology-directed repair in somatic cells such as cardiomyocytes. In summary, RNA-targeted therapies (e.g. apo(a)-ASO and PCSK9-siRNA) are now in large-scale clinical outcome trials and will most likely become a novel effective and safe therapeutic option for CVD in the near future. MicroRNA-modulating, epigenetic, and gene therapies are tested in early clinical studies for CVD. CRISPR-Cas-mediated genome editing is highly effective in stem cells, but major challenges are remaining in somatic cells, however, this field is rapidly advancing.

Background The 2017 Japan Atherosclerosis Society (JAS) familial hypercholesterolemia (FH) criteria adopts a cut off value of ≥9 mm of Achilles tendon thickness (ATT) detected by X-ray as one of the three key items. This threshold was determined based on an old data assessing ATT of 36 non-FH individuals published in 1977. Although the specificity of this clinical criteria is extremely high due to a strict threshold, there are substantial number of patients with FH whose ATT Methods The individuals (male/female = 486/501) with full assessments of genetic analyses for FH-genes (LDLR, and PCSK9), serum lipids, and ATT detected by X-ray at Kanazawa University Hospital and National Cerebral and Cardiovascular Center Research Institute were included in this study. Receiver operating characteristic (ROC) analyses were performed to determine a better cut off point of ATT predicting a pathogenic mutation of FH. Results ROC analyses revealed the best cut off values of ATT as 7.6 mm for male, and 7.0 mm for female with the sensitivities and specificities of 0.83 and 0.83 for male and 0.86 and 0.85 for female, respectively. If the thresholds of ATT of 8.0/7.5 mm and 7.5/7.0 mm were applied to diagnose of male/female FH, the sensitivities/specificities predicting a pathogenic mutation of FH by the 2017 JAS FH clinical criteria would be 0.82/0.90 and 0.85/0.88, respectively. Conclusions These results suggest that the cut-off value of ATT detected by X-ray is obviously lower than 9.0 mm adopted by the 2017 JAS FH clinical criteria. Funding Acknowledgement Type of funding sources: None.

Background The 2019 ESC dyslipidaemia guidelines recommend a more intense LDL-cholesterol (LDL-C) reduction for patients with coronary artery disease (CAD). As a result the need for PCSK9 inhibitors (PCSK9i) is projected to increase significantly, with a concomitant substantial impact on treatment cost. Adding the novel lipid lowering agent bempedoic acid (BA) to established oral lipid lowering therapy could provide an affordable alternative to PCSK9i to attain the LDL-C treatment target, particularly in patients with statin intolerance. Aims To quantify the target populations for BA and PCSK9i as well as the related treatment costs to achieve the LDL-C target of the 2019 ESC dyslipidaemia guidelines assuming addition of BA to lipid lowering medication. Methods We included 1922 patients (mean age 69.3 years) with angiographically documented CAD from an observational cohort study ongoing since 2015. A Monte Carlo simulation incorporating an algorithm for intensification of lipid lowering medication was applied in order to achieve the LDL-C treatment goal of Results Figure 1 displays the baseline LDL-C distribution (median LDL-C 86.0 mg/dL) and lipid lowering medications. The need for PCSK9i in scenario 1 would be 41.4% for a moderate and 46.1% for a high rate of statin intolerance. Addition of BA in scenario 2 would reduce the need for PCSK9i to 25.3% and 29.4%. Figure 2 displays the LDL-C distributions and lipid lowering medications for scenarios 1 and 2 assuming a moderate rate of statin intolerance. Use of BA would lower the annual overall treatment cost incurred through use of PCSK9i ± BA per 1,000,000 CAD patients by 13.3% and 10.5%. The cost per prevented event in the entire cohort would be lower (−5.0 and −6.3%), however at the price of less prevented cardiovascular events (−8.7% and −4.5%). Amongst patients with full statin intolerance, the cost per prevented event would be reduced (−6.8% for both rates of statin intolerance) whilst more cardiovascular events would be prevented (+7.5% and +6.9%). Conclusion Use of BA in patients with CAD would reduce the need for PCSK9i as well as the overall treatment cost for add-on lipid lowering therapy. The subpopulation of patients with full statin intolerance might profit particularly. Funding Acknowledgement Type of funding sources: None. Figure 1. Baseline LDL-C distribution and LLMFigure 2. LDL-C and LLM after LLM intensification

Background The role of proprotein convertase subtilisin/kexin type 9 (PCSK9) in predicting major adverse cardiovascular events (MACEs) in Non-ST elevation myocardial infarction (NSTEMI) patients is still an open question and the PCSK9 concentration of clinical usefulness remains unknown in guiding treatment. Purpose To explore the role of PCSK9 in predicting major adverse cardiovascular events (MACEs) in Non-ST elevation myocardial infarction patients. Methods 272 patients with NSTEMI were included in our study, all patients received PCI therapy after admission. Patients were followed up for 1 year and MACEs were recored. Their baseline plasma PCSK9 levels were determined by ELISA. Patients were divided into high, medium and low PCSK9 groups and the associations of PCSK9 with other biomarkers and MACEs were evaluated. Results The results showed that PCSK9 levels was related to levels of lipoproteins, high-sensitivity C-reactive protein (r=0.162, P=0.008), platelet volume distribution width (r=0.299, P Conclusions In a NSTEMI setting, the concentration of PCSK9 is associated with hypercoagulability and hyper-inflammation. High levels of PCSK9 independently predict future MACEs in NSTEMI patients, particularly those classified by the GRACE score as high risk. Funding Acknowledgement Type of funding sources: None.

Introduction Inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9) is a novel treatment for hypercholesterolaemia; recent studies evidenced that PCSK9i reduces cardiovascular diseases and risk of atherosclerosis. Evolocumab, a PCSK9i, reduced the risk of cardiovascular events in patients with atherosclerotic cardiovascular diseases when added to maximally tolerated statin therapy (± ezetimibe), and recent data from the ODYSSEY OUTCOMES trial indicate that alirocumab added to maximally tolerated statin therapy (± other lipid-lowering drugs) reduces the risk of cardiovascular events in patients with a recent acute coronary syndrome. Purpose Considering the expression of PCSK9 in heart tissues and cardiomyocytes, we aimed to study for the first time the putative cellular effects of evolocumab in human cardiomyocytes incubated with doxorubicin, trastuzumab, their sequential treatments Methods Human fetal cardiomyocytes (HFC cell line) were exposed to subclinical concentration of doxorubicin, trastuzumab, sequential treatment of both (all 100 nM), alone or in combination with evolocumab (50 nM) for 48h. After the incubation period, we performed the following tests: determination of cell viability, through analysis of mitochondrial dehydrogenase activity, study of lipid peroxidation (quantifying cellular Malondialdehyde and 4-hydroxynonenal), intracellular Ca2+ homeostasis. Moreover, pro-inflammatory studied were also performed (activation of NLRP3 inflammasome; expression of TLR4/MyD88; mTORC1 Fox01/3a; transcriptional activation of p65/NF-κB and secretion of cytokines involved in cardiotoxicity (Interleukins 1β, 8, 6). Results Evolocumab co-incubated with doxorubicin alone or in sequence with trastuzumab exerts cardioprotective effects, enhancing cell viability of 35–43% compared to untreated cells (p Conclusion PCSK9 inhibitor evolocumab exerts direct cellular effects in cardiomyocytes during doxorubicin and trastuzumab exposure with anti-inflammatory effects. These results indicated that evolocumab should be studied in cancer-bearing mice treated with anthracyclines and HER2-blocking agents in order to reduces cardiovascular events. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Ministero della Salute, Ricerca Corrente Project

Background The reasons why patients are treated or not with PCSK9 inhibitors (PCSK9i) are incompletely understood. In Germany, access to PCSK9i is limited by local regulations and many high-risk cardiovascular patients do not receive these therapies. The PERI-DYS study aims to describe and compare two groups of dyslipidaemia patients at very high CV risk: those treated with PCSK9i compared with patients qualifying for but not treated with PCSK9i. Methods Observational study with up to 2000 consented patients, documented mainly by office-based cardiologists or physicians in lipid ambulances with data extracted from patient charts. Lipid lowering treatment (LLT) at enrolment includes ongoing PCSK9i use, newly initiated PCSK9i, statins, ezetimibe, and lipoprotein apheresis. Patients are followed for up to 3 years, with visits every 6±2 months to record LLT (drugs, dosing), other CV medications, lipid and glucose values, blood pressure, clinical events (major adverse cardiac and cerebrovascular events) and adverse drug reactions. Results As of 05 March 2021, 1488 patients have been enrolled across 70 sites. The majority of patients (91.5%) had heterozygous familial or non-familial hypercholesterolemia or mixed dyslipidaemia. At enrolment, 49.4% of patients were receiving PCSK9i (35.4% ongoing and 14.0% newly treated). Among PCSK9i users, the majority were receiving evolocumab 140 mg (n=567, 38.1% of all enrolled patients). There were no major differences in demographics and non-lipid lowering medication, with the exception of more females in the PCSK9i group. The estimated untreated LDL-C based on “back-calculation” was higher in patients who were on ongoing PCSK9i therapy than in those not on PCSK9i or newly treated with PCSK9i (Table 1). Physician-reported statin intolerance was much more common in the two PCSK9i groups compared with the non-PCSK9i group (67% versus 14%). Patients in the PCSK9i groups received fewer concomitant statins. Mean on-treatment total cholesterol and LDL-C were considerably lower in patients who were on ongoing PCSK9i compared to non-PCSK9i. Overall, nicotinic acid, fibrates, cholestagel, and omega-3 fatty acids were rarely used (data not shown). Conclusions Patients treated with PCSK9i and those qualifying for but not treated with PCSK9i had similar baseline characteristics, but the former had higher estimated untreated LDL-C values and a higher rate of statin intolerance. Ongoing follow-up will determine the prognostic importance of these findings. Funding Acknowledgement Type of funding sources: Private company. Main funding source(s): Amgen GmbH Germany

Aims The aim of this study was to assess the impact and cost-effectiveness of offering population genomic screening to all young adults in Australia to detect heterozygous familial hypercholesterolaemia (FH). Methods and results We designed a decision analytic Markov model to compare the current standard of care for heterozygous FH diagnosis in Australia (opportunistic cholesterol screening and genetic cascade testing) with the alternate strategy of population genomic screening of adults aged 18–40 years to detect pathogenic variants in the LDLR/APOB/PCSK9 genes. We used a validated cost-adaptation method to adapt findings to eight high-income countries. The model captured coronary heart disease (CHD) morbidity/mortality over a lifetime horizon, from healthcare and societal perspectives. Risk of CHD, treatment effects, prevalence, and healthcare costs were estimated from published studies. Outcomes included quality-adjusted life years (QALYs), costs and incremental cost-effectiveness ratio (ICER), discounted 5% annually. Sensitivity analyses were undertaken to explore the impact of key input parameters on the robustness of the model. Over the lifetime of the population (4 167 768 men; 4 129 961 women), the model estimated a gain of 33 488years of life lived and 51 790 QALYs due to CHD prevention. Population genomic screening for FH would be cost-effective from a healthcare perspective if the per-test cost was ≤AU$250, yielding an ICER of Conclusion Based on our model, offering population genomic screening to all young adults for FH could be cost-effective, at testing costs that are feasible.

Background Recent ESC/EAS Guidelines for the management of dyslipidemia stated that the treatment goal of LDL cholesterol (LDL-C) in very high-risk patients is less than 55mg/dl. PCSK9 inhibitors in addition to strong statins could be a useful strategy for rapid and aggressive lowering of LDL-C. However, the feasibility and safety of early initiation of a PCSK9 inhibitor for AMI patients undergoing primary PCI remain unclear. Objectives We examined the effects of early initiation of a PCSK9 inhibitor, evolocumab, on lipid profile and inflammatory markers and its safety in AMI patients undergoing primary PCI. Methods This study is a single center, randomized, controlled trial involving 102 patients hospitalized for AMI. The patients were randomly assigned 1:1 to the evolocumab group and the control group. Evolocumab (140 mg) was subcutaneously injected within 24 hours after PCI and then every two weeks. All patients received pitavastatin (2mg/day) in addition to the allocated treatment. The primary endpoints were changes in lipid profile and inflammatory markers from baseline to 4 weeks. Results 102 patients were enrolled between October 2017 and December 2019. 89 patients were ST-segment elevation myocardial infarction (STEMI), 13 patients were non-STEMI. Primary PCI was successfully performed in all patients. 76 patients were statin-naïve. 2 patients were excluded from analyses because they died severe heart failure in acute phase. Finally, 100 patients (evolocumab; n=51 and control; n=49) were analyzed. Baseline LDL-C was 121.6±30.3 mg/dl in the evolocumab group and 124.7±33.6 mg in the control group. Change in LDL-C from the baseline to 4 weeks was −92.4±32.4 mg/dl (−75%) in the evolocumab group and −44.8±32.1 mg/dl (−33.1%) in the control group (mean difference; 47.6mg/dl, 95% CI; 34.8 to 60.4 mg/dl, p Conclusion In patients with AMI undergoing primary PCI, early initiation of evolocumab rapidly reduced LDL-C without no adverse event, and achieved LDL-C Funding Acknowledgement Type of funding source: None

Background and purpose In acute coronary syndrome (ACS) patients, major cardiovascular events during follow-up were equally attributable to recurrence at the site of culprit lesions and to non-culprit lesions, and mostly occurred within one year. Recent studies showed that Proprotein convertase subtilisin kexin type 9 (PCSK9) inhibitor significantly reduces the risk of cardiovascular events. However, the rigorous effects of PCSK9 inhibitor in patients with ACS, especially for the stabilization of plaque in the non-culprit lesions has not been identified. The purpose of this study was to evaluate the efficacy of a PCSK9 inhibitor for the plaque stabilization in the non-culprit lesion of ACS patients. Methods We retrospectively analyzed the 10 ACS patients (STEMI:5 cases, NSTEMI:1 case, and UAP:4 cases) who had non-culprit lesions and were injected with a PCSK9 inhibitor. We analyzed 11 non-culprit lesions before and after PSCK9 injections by using a near-infrared intravascular ultrasonography (NIRS-IVUS). Results The follow up intervals were 229.4±82.9 days. Major cardiovascular events did not occur in all patients. The serum low-density lipoprotein (LDL) cholesterol levels were significantly decreased form 128±36.7 mg/dl to 26.7±7.4 mg/dl (P Conclusion This study suggested that the plaque morphology can be altered by the aggressive lipid lowering therapy by using a PCSK9 inhibitor, and NIRS-IVUS can detect these plaque stabilizations of non-culprit lesion in ACS patients. The authors have no financial conflicts of interest to disclose concerning the presentation. Figure 1 Funding Acknowledgement Type of funding source: None

Background PCSK9 antibodies are novel potent and expansive lipid lowering agents that demonstrated clinical benefit in high risk patients. We hypothesized that optimization of dose and administration schedule, ideally adapted to the target population, could reduce costs while maintaining the clinical benefit. Objective To explore the relationship between LDL-Cholesterol (LDL-C) decrease by anti-PCSK9 monoclonal antibodies and several covariates such as drug dose, administration schedule, baseline LDL-C, population and statins. Methods We performed systematic review, meta-analysis and meta-regression of randomized controlled trials that compared alirocumab or evolocumab to placebo or no treatment and reported LDL-C decrease, with a minimum follow-up of 12 weeks and with a sample size of 30 patients or more. Electronic searches of MEDLINE, EMBASE, CENTRAL and ClinicalTrials.gov from inception to March 2019. We evaluated the quality of included studies and extracted aggregate data. We used random effect models and multivariate multilevel meta-regression to explore factors influencing LDL-C decrease. All analyses were performed with R. Results From 1479 references identified and screened on title/abstract, the full texts of 72 articles were screened. We included 32 studies (31 references.) Anti-PCSK9 mAbs decreased LDL-C by 53%, 95% CI (−56% to −50%), with no significant difference between the two drugs (p=0.07). In univariate meta-regressions, higher baseline LDL-C level, monthly administration, higher percentage of patients with high-dose statins were associated with a lower LDL-C decrease (p (p Conclusion Alirocumab and evolocumab showed substantial LDL-C reductions in clinical trials, without significant differences in their biological efficacies. A higher baseline LDL-C, higher intensity of statin co-treatment and a lower dose seemed to negatively influence LDL-C decrease. Funding Acknowledgement Type of funding source: None

Aim PCSK9 has been recently associated with a higher rate of calcification in hypercholesterolemic, diabetes and CKD patients. The aim of this study was thus to investigate the role of PCSK9 in VC process, under uremic condition, both on in vivo and in vitro experimental settings. Methods Sprauge Dawley rats were fed a standard diet (SD, n=11) or uremic diet (UD, n=11) for 6 weeks. Calcium crystals in aortas were visualized by von Kossa staining and quantified by a colorimetric assay and plasma total cholesterol determined. Control and PCSK9-overexpressing smooth muscle cells (SMCsPCSK9) were cultured with 2.5% FCS ± Pi for 7 days. Hydroxyapatite deposition by SMCs was measured by a colorimetric assay. The number and the content of pro-calcific extracellular vesicles (EVs) budding from the cells were determined. Results The hyperphosphatemia secondary to CKD lead to rat aortic calcification (+7.3-fold) and a significant increase in TC and PCSK9 levels (+1.4 and +2.7-fold, respectively). Higher expression of PCSK9 was also observed in kidney (+4.8-fold) and liver (+1.5-fold). SMCsPCSK9 showed higher extracellular calcium deposition (+1.4-fold) in response to Pi and increase EVs production (+7-fold). The incubation of control cells with recombinant PCSK9 did not induce extracellular calcium deposition. Conclusions Our study suggest a positive effect of intracellular PCSK9 on vascular calcification in CKD condition. Pro-calcific budding of EVs seems one of the possible mediators of this process. PCSK9 and calcification Funding Acknowledgement Type of funding source: None

Background The risk of atherosclerosis and cardiovascular disease (CVD) is very high in SLE. This is both a clinical problem and of interest in studies of immunity in these conditions. LDL-levels are increased by Proprotein convertase subtilisin kexin 9 (PCSK9) which targets the LDL-receptor (LDLR). We recently reported that PCSK9 ameliorates dendritic cell (DC) activation by oxidized LDL (OxLDL), which is abundant in atherosclerotic plaques and also raised and associated with cardiovascular disease (CVD) in SLE. We here investigate the role of PCSK9 in SLE. Methods PCSK9-levels were determined by ELISA among SLE patients (n=109) and age- and sex-matched population-based controls (n=91). Common carotid intima-media thickness (IMT) and plaque occurrence were determined by B-mode ultrasound. Plaques were graded by echogenicity. Human peripheral blood monocytes from SLE patients or controls were differentiated into DCs. Effects of PCSK9 and its inhibition by silencing were studied. Results PCSK9-levels were non-significantly higher among SLE-patients as compared to controls but associated significantly with SLE disease activity, as determined by SLAM (0.020) or SLEDAI (0.0178). There was no association between PCSK9-levels and atherosclerosis as determined by IMT, prevalence of plaques or echolucent (potentially vulnerable) plaques. PCSK9 levels were significantly associated with CVD among SLE-patients. OxLDL induced PCSK9 in DCs and DC-maturation with increased expression of CD86 and HLA-DR. The effects were significantly stronger in DC from SLE patients than from controls. Silencing of PCSK9 abolished OxLDL-induced DC-maturation. Conclusions PCSK9 is associated with disease activity in SLE. One underlying cause could be OxLDL, promoting DC-activation which depends on PCSK9. OxLDL induces PCSK9, an effect which is higher among SLE-patients. PCSK9 could play an unexpected immunological role in SLE. Funding Acknowledgement Type of funding source: Private company. Main funding source(s): Amgen; Swedish Heart and Lung Foundation

Background The proprotein convertase subtilisin/kexin type 9 inhibitor (PCSK9-I), evolocumab, reduced the risk of cardiovascular event in patients with peripheral artery disease in FOURIER trial. However, the effects of evolocumab on favorable limb outcomes in patients with critical limb ischemia (CLI) is still unclear. Purpose The aim of this study was to evaluate the impacts of evolocumab on favorable limb outcomes and lipid profile in patients with CLI. Methods This was a single center, prospective observational study. A total of 39 patients with CLI were enrolled between November 2016 to May 2019. The subjects were divided into 2 groups based on evolocumab administration: evolocumab-treated group: E group (mean 69.4±11.7 years, n=14) and evolocumab non-treated group: Non-E group (mean 74.0±8.8 years, n=25). Baseline characteristics were assessed at admission. Lipid profile was evaluated at admission, 1, 3, 6, 12 and 18 months. The primary outcome was defined 18-month amputation-free survival (AFS). The secondary outcomes were defined 18-month overall survival (OS) and wound-free limb salvage. Mean follow-up period was 18±11 months. Results The patients in E group had greater reduction in levels of LDL cholesterol and non-HDL cholesterol than those in Non-E group over time. The reduction in MDA-LDL level was maintained at 1, 3, 6, 12 months, respectively. The 18-month AFS rate in the E-group was significantly higher than those in the Non-E group (log-rank p=0.02). The patients receiving evolocumab had a lower hazard regarding AFS (hazard ratio, 0.12; 95% confidence interval, 0.02–0.94; P=0.043) and a higher proportion of wound-free limb salvage at 12 months (E group [92%] vs Non-E group [57%], P=0.034) and 18 months (92% vs 52%, P=0.03). Otherwise, evolocumab administration was not associated with 18-month OS (log-rank p=0.053). Conclusions Evolocumab administration may be associated with the favorable outcome of 18-month AFS in the patients with CLI. Additionally, long-term administration of evolocumab over 12 months may improve wound-free limb salvage. Effects of evolocumab on limb outcomes Funding Acknowledgement Type of funding source: None

Introduction Inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged as a novel therapy to treat hypercholesterolaemia and related cardiovascular diseases. Evolocumab, a PCSK9 inhibitor, reduced the risk of cardiovascular events in patients with atherosclerotic cardiovascular diseases when added to maximally tolerated statin therapy (± ezetimibe), and recent data from the ODYSSEY OUTCOMES trial indicate that alirocumab added to maximally tolerated statin therapy (± other lipid-lowering drugs) reduces the risk of cardiovascular events in patients with a recent acute coronary syndrome. Purpose Considering the expression of PCSK9 in heart tissue, we aimed to study for the first time the direct biochemical effects of evolocumab in cardiomyocytes during exposure to doxorubicin, trastuzumab, their sequential treatments, and immune checkpoint inhibitor ipilmumab. Methods Human fetal cardiomyocytes (HFC cell line) were exposed to subclinical concentration of doxorubicin, trastuzumab, sequential treatment of both ( all 100 nM), alone or in combination with evolocumab (50 nM) for 48h. In another experiment, in co-coltures of human fetal cardiomyocytes and lymphocytes, we incubated ipilimumab (200 nM) alone or in combination with evolocumab for 48h. After the incubation period, we performed the following tests: determination of cell viability, through analysis of mitochondrial dehydrogenase activity, study of lipid peroxidation (quantifying cellular Malondialdehyde and 4-hydroxynonenal), intracellular Ca2+ homeostasis. Moreover, pro-inflammatory studied were also performed (activation of NLRP3 inflammasome; expression of TLR4/MyD88; mTORC1 Fox01/3a; transcriptional activation of p65/NF-κB and secretion of cytokines involved in cardiotoxicity (Interleukins 1β, 8, 6). Results Evolocumab co-incubated with doxorubicin alone or in sequence with trastuzumab exerts cardioprotective effects, enhancing cell viability of 35–43% compared to untreated cells (p Conclusion We demonstrated, for the firts time, that PCSK9 inhibitor evolocumab exerts direct effects in cardiomyocytes during doxorubicin, trastuzumab and ipilimumab mediated cardiotoxicity turning on a new light on its possible use in the management of the cardiotoxic effects of antineoplastic drugs in cancer patients Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): This work was funded by the “Ricerca Corrente” grant from the Italian Ministry of Health

Introduction Familial hypercholesterolemia (FH) is an autosomal dominant disease that leads to premature cardiovascular disease (CAD). The availability of ab-PCSK9 has changed the approach to therapy. Purpose To evaluate the relationship between different types of mutations in LDLR gene and response to ab-PCSK9. Methods 73 FH patients, 33 women and 40 men (53.9±13. yrs), in primary prevention (N=46) and secondary prevention (N=27), were recruited. This sample included patients with mutations in LDLR gene: heterozygotes for missense mutations (N=31), for null mutations (N=31), compound heterozygotes or homozygotes (N=11). At baseline, the whole sample had a maximally tolerated lipid lowering therapy (MT-LLT) without ab-PCSK9; 16 patients had MT-LLTs intolerance. After 160 days with ab-PCSK9 therapy we evaluated the achievement of a goal (LDL-C Results After 160 days of therapy with ab-PCSK9 (45 patients on Alirocumab, 28 patients on Evolocumab) and MT-LLT, 29/73 patients (39.7%) of the whole sample achieve the goal of LDL-C. Of them 14/29 (48.2%) were in primary prevention, 15/29 (51.7%) in secondary prevention, no difference in achievement of the goal. We then evaluated the percent of patients achieving the goal of LDL-C: 15/31 (48.3%) patients with missense mutation and 14/31 (45.1%) patients with null mutation, no significant difference among groups; 0/11 compound heterozygotes or homozygotes; 3/16 (18.7%) MT-LLTs intolerance. The other main cardiovascular risk factors did not influence of the achievement the goal of LDL cholesterol. Conclusions Lack of correlation between type of mutation in heterozygous FH patients and ab-PCSK9 therapy response; response was significantly poorest in patients with compound heterozygosis or homozygosis mutation as compared to heterozygotes; the intolerance to MT-LLT was significant in the achievement of the goal of LDL-C. Different between guideline 2016 vs 2019 Funding Acknowledgement Type of funding source: None

Background LDL cholesterol (LDL-C) is a well-established risk factor for cardiovascular disease. PCSK9 binds LDL receptors, targeting them for degradation. The dosing intervals for currently available PCSK9 monoclonal antibodies are once every 2 or 4 weeks. Tafolecimab, a novel recombinant human PCSK9 monoclonal antibody, was found to have higher affinity with PCSK9 and show longer LDL-C reduction compared to evolocumab in preclinical studies. Purposes The objectives for the SAD and MAD studies were to investigate the safety and efficacy of tafolecimab and explore the optimal dosing schedule. Methods The phase 1 study was a randomized, placebo-controlled, double-blind, single-ascending dose study (SAD) in Chinese healthy subjects, who were randomized 3:1 to tafolecimab and placebo (n=58). SAD subjects received tafolecimab subcutaneously at 25/75/150/300/450/600mg, or intravenously at 75/450mg, monitored up to day 84. The phase 2 study was a randomized, double-blind, placebo-controlled, repeated-dosing, multiple ascending dose (MAD) study in patients with hypercholesterolemia, who were randomized 4:1 to tafolecimab and placebo (n=60). MAD subjects received tafolecimab subcutaneously at 75/140mg every 2 weeks, 300/420mg every 4weeks, 450/600mg every 6 weeks up to day 84 or 98 with 3 months follow-up. Results In the SAD, the maximum mean reduction in LDL-C ranged from 52.2% to 72.1% and was achieved as early as 5 days (figure 1a). The duration of LDL-C reduction was tafolecimab dose dependent. In the MAD, the mean LDL-C concentrations were reduced by tafolecimab for each dose at 12 weeks relative to baseline (ranging from 54.30% to 72.26%; p Both healthy and hypercholesterolemia subjects are generally tolerable to tafolecimab. Reported treatment-emergent adverse events (TEAEs) were: tafolecimab 23 (52.3%) vs. placebo 8 (57.1%); tafolecimab 34 (70.8%) vs. placebo 9 (75.0%) in the SAD and MAD respectively. There were no serious TEAEs or events leading to death or treatment discontinuation in both SAD and MAD. Conclusions Tafolecimab was well tolerated in both healthy and hypercholesterolemia in Chinese subjects, and improved lipid profile including LDL-C, Lp(a) and other lipids. The sustained effects on LDL-C suggests the potential of tafolecimab as a long-lasting PCSK9 inhibitor with dosing interval of 6–8 weeks or beyond. Figure 1. LDL-C: Percent change from baseline Funding Acknowledgement Type of funding source: Private company. Main funding source(s): Innovent Biologics (Suzhou), China

Introduction The recently updated ESC guidelines on the management of dyslipidaemias recommend a more intense LDL-cholesterol (LDL-C) reduction. For patients with atherosclerotic cardiovascular disease (ASCVD) the LDL-C goal has been revised to ≤55 mg/dl with a concomitant class IA upgrade for cost intensive PCSK9 inhibitors. Purpose We aim to quantify the need for PCSK9 inhibitors to achieve the revised LDL-C target compared to former ESC recommendations in ASCVD patients Methods We included all patients with ASCVD (angiographically documented coronary artery disease, history of peripheral artery disease or stroke) from an observational cohort study ongoing since 2015. A simulation treatment algorithm adding sequentially a high intensity statin, ezetimibe and a PCSK9 inhibitor in case of a missed treatment target was applied with consideration of both partial and total statin intolerance. The need for PCSK9 inhibitors was calculated for 3 recommendations: 1. LDL-C treatment target ≤55 mg/dl (ESC 2019 Guidelines), 2. LDL-C treatment target ≤70 mg/dl (ESC 2016 Guidelines) and 3. risk-based use of PCSK9 inhibitors restricted to patients with a residual LDL-C >140 mg/dl or >100 mg/dl with clinical/angiographic risk factors (ESC consensus update 2017). Results We included 1936 patients (mean age 69 years, 74% male). Median LDL-C at inclusion was 86 mg/dl, with 60% of patients taking lipid lowering medication (55% statin only, 4% statin + ezetimibe, 1% ezetimibe only). Table 1 shows the distribution of medications required to meet recommendations 1–3. After simulated stepwise intensification of lipid lowering therapy 99% of patients achieved the revised LDL-C target of ≤55 mg/dl, with a need of 23.5% for a PCSK9 inhibitor. For the former LDL-C target of ≤70 mg/dl the need for PCSK9 inhibitors was 10.5%. Restricting the use of PCSK9 inhibitors to the highest risk patients according to the ESC 2017 consensus statement reduced the need for PCSK9 inhibition to only 1.4% with slightly fewer patients achieving their LDL-C target (78% for ≤55 mg/dl and 91% for ≤70 mg/dl respectively). Conclusion The revised LDL-C treatment goals substantially increase the projected need for PCSK9 inhibitors with an unclear health economic impact. Identification of ASCVD patients with a reasonable benefit/cost-ratio of PCSK9 inhibition remains to be investigated urgently. Funding Acknowledgement Type of funding source: None

Background Some STEMI patients still suffers adverse outcomes though they undergo optimal management, including primary PCI. Plasma PCSK9 is deemed to have a role in that residual risk. Our knowledge on the association between plasma PCSK9 level and MACCE in STEMI is still limited. Purpose The aim of this study is to evaluate the association between plasma PCSK9 level during admission with MACCE in STEMI who undergo primary PCI. Methods This was a prospective observational study, recruited 239 patients with STEMI who were treated with primary PCI and assayed for PCSK9 level using ELISA technique. MACCE and other supportive data were taken from the medical records and telephone follow-up. Results There were 28 study participants who experienced MACCE in 30 days. However, survival analysis did not show a significant association between plasma PCSK9 level and MACCE in 30 days. The hazard ratio for MACCE between the third tertile and the second tertile of plasma PCSK9 level was 1.466 (95%CI 0.579-3.714) and between the first tertile and the second tertile was 1.257 (95%CI 0.496-3.185). Conclusion Plasma PCSK9 levels was not associated with MACCE in STEMI patients treated with primary PCI.

Aims: Low-density lipoprotein (LDL) particles cause atherosclerotic cardiovascular disease (ASCVD) through their retention, modification, and accumulation within the arterial intima. High plasma concentrations of LDL drive this disease, but LDL quality may also contribute. Here, we focused on the intrinsic propensity of LDL to aggregate upon modification. We examined whether inter-individual differences in this quality are linked with LDL lipid composition and coronary artery disease (CAD) death, and basic mechanisms for plaque growth and destabilization. Methods and results: We developed a novel, reproducible method to assess the susceptibility of LDL particles to aggregate during lipolysis induced ex vivo by human recombinant secretory sphingomyelinase. Among patients with an established CAD, we found that the presence of aggregation-prone LDL was predictive of future cardiovascular deaths, independently of conventional risk factors. Aggregation-prone LDL contained more sphingolipids and less phosphatidylcholines than did aggregation-resistant LDL. Three interventions in animal models to rationally alter LDL composition lowered its susceptibility to aggregate and slowed atherosclerosis. Similar compositional changes induced in humans by PCSK9 inhibition or healthy diet also lowered LDL aggregation susceptibility. Aggregated LDL in vitro activated macrophages and T cells, two key cell types involved in plaque progression and rupture. Conclusion: Our results identify the susceptibility of LDL to aggregate as a novel measurable and modifiable factor in the progression of human ASCVD.

Aims Despite patient reports of neurocognitive disorders with lipid-lowering treatments (LLTs), large clinical trials have found no significant association between neurocognitive disorders and LLTs. We assessed incidence of neurocognitive treatment-emergent adverse events (TEAEs) from 14 Phase 2 and 3 trials of the proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitor alirocumab. Methods and results Patients (most on background maximally tolerated statin) received alirocumab 75/150 mg every 2 weeks (n = 3340; 4029 patient-years of exposure), placebo (n = 1276), or ezetimibe (n = 618). Data were pooled by the control used. Neurocognitive TEAEs were reported by 22 (0.9%) alirocumab-treated patients vs. 9 (0.7%) with placebo in placebo-controlled trials [hazard ratio (HR) 1.24, 95% confidence interval (CI) 0.57–2.68] and 10 (1.2%) with alirocumab vs. 8 (1.3%) with ezetimibe in ezetimibe-controlled trials (HR 0.81, 95% CI 0.32–2.08). Rates of neurocognitive TEAEs were similar in patients receiving alirocumab with LDL cholesterol (LDL-C) levels

Aims Migration of monocytes into the arterial wall contributes to arterial inflammation and atherosclerosis progression. Since elevated low-density lipoprotein cholesterol (LDL-C) levels have been associated with activation of plasma monocytes, intensive LDL-C lowering may reverse these pro-inflammatory changes. Using proprotein convertase subtilisin/kexin type 9 (PCSK9) monoclonal antibodies (mAbs) which selectively reduce LDL-C, we studied the impact of LDL-C lowering on monocyte phenotype and function in patients with familial hypercholesterolaemia (FH) not using statins due to statin-associated muscle symptoms. Methods and results We assessed monocyte phenotype and function using flow cytometry and a trans-endothelial migration assay in FH patients (n = 22: LDL 6.8 +/- 1.9 mmol/L) and healthy controls (n = 18, LDL 2.9 +/- 0.8 mmol/L). Monocyte chemokine receptor (CCR) 2 expression was approximaterly three-fold higher in FH patients compared with controls. C-C chemokine receptor type 2 (CCR2) expression correlated significantly with plasma LDL-C levels (r = 0.709) and was positively associated with intracellular lipid accumulation. Monocytes from FH patients also displayed enhanced migratory capacity ex vivo. After 24 weeks of PCSK9 mAb treatment (n = 17), plasma LDL-C was reduced by 49%, which coincided with reduced intracellular lipid accumulation and reduced CCR2 expression. Functional relevance was substantiated by the reversal of enhanced migratory capacity of monocytes following PCSK9 mAb therapy. Conclusions Monocytes of FH patients have a pro-inflammatory phenotype, which is dampened by LDL-C lowering by PCSK9 mAb therapy. LDL-C lowering was paralleled by reduced intracellular lipid accumulation, suggesting that LDL-C lowering itself is associated with anti-inflammatory effects on circulating monocytes

Introduction Platelets interact with circulating CD34+ progenitor cells inducing their differentiation into endothelial progenitor cells (EPCs) and further promoting EPC maturation into endothelial cells, an important step for endothelial regeneration and angiogenesis. PSCK9 is a serine protease that may not only play a crucial role in binding to the low density lipoprotein receptor (LDL-R) increasing its endosomal and lysosomal degradation thus inhibiting its recycling to the cell surface, leading to increasing LDL-cholesterol levels in plasma, but may also exhibit several, independent on LDL-R, activities on a plethora of cell types, including platelets. Purpose We investigated whether monoclonal antibodies (mabs) against PCSK9 could affect platelet interaction with CD34+ cells as well as their differentiation into EPCs, in patients with Familial Hypercholestorelemia (FH). Patients and methods Patients with FH (n=11, 8 men and 3 women, mean age 53±10 years) being under statin/ezetimibe therapy (40mg statin/ 10mg ezetimibe) participated in the study. Patients exhibited resisting high LDL-cholesterol levels, therefore following the existing guidelines, they received mab against PCSK9 (7 evolocumab, 140mg/ml and 4 alirocumab, 150mg/ml) every two weeks. Blood samples were drawn before and after 4 doses of the antibody i.e. after two months (Follow-up). In addition to the patients' serum lipid profile, the endothelial phenotype of CD34+ (membrane expression levels of KDR which is a receptor for VEGF and an endothelial phenotype marker) on CD34+ cells (CD34+-KDR+ cells) and the formation of platelet-CD34+ (CD61+-CD34+) and platelet-KDR+ (CD61+-KDR+) conjugates were also determined by flow cytometry on whole blood after dual labelling with anti-VEGFR-R2/KDR-PE, anti-CD61-PerCP and anti-CD34-FITC. The results were expressed as the %gated of CD34+-KDR+ cells, as well as CD61+-CD34+ and CD61+-KDR+ conjugates. Results The baseline LDL-cholesterol levels were significantly reduced from 172±43 mg/dl to 51±17 at follow-up, p=0.0001. Importantly, the formation of CD61+-CD34+ and CD61+-KDR+ conjugates were increased from baseline to follow-up (from 1.39±1.3 to 2.23±2.8, p=0.037) and (from 2.91±4.2 to 5.92±6.01, p=0.014), respectively. The membrane expression of KDR on CD34+ cells was also increased from 0.79±0.9 to 1.36±0.90, p=0.042, which reflects the increase in the CD34+ endothelial phenotype. Conclusion We show for the first time that the mabs against PCSK9, significantly increase the CD34+ endothelial phenotype in FH patients, which may at least partially attributed to the increase in the platelet interaction with CD34+ cells (platelet-CD34+ conjugates). The underlying mechanisms as well as the consequences of this effect at the clinical level for these patients are under investigation.

Background PCSK9 inhibitors have emerged as an effective lipid-lowering approach. Although the results of available studies suggest a positive association of plasma PCSK9 levels with glycemic parameters and risk of DM, the effects of PCSK9 inhibitors on glucose intolerance and insulin resistance as the key features of DM remain unclear. Purpose The present study was directed to determine effect of vaccine-mediated PCSK9 inhibition on glucose intolerance and insulin resistance in experimental diabetic rats. Methods Nanoliposomal vaccine composing from PCSK9-linked nanoliposome particles mixed in Alum adjuvant was subcutaneously injected four times with bi-weekly intervals in Wistar-Albino rats. Two weeks after the last immunization, vaccinated and non-vaccinated rats were subjected to diabetes experiment induced by single intraperitoneal injection of streptozotocin (STZ). One week after STZ injection, glucose tolerance ability of each animal was evaluated by using oral glucose tolerance test (OGTT) on the overnight fasted rats with glucose dose at 2 g/kg. Two weeks after STZ injection, insulin tolerance test (ITT) viaintraperitoneal injection of insulin (0.8 U/kg) was performed to determine the measure of peripheral utilization of glucose. The plasma concentrations of total cholesterol (TC), LDL-C, HDL-C, and TG were measured. Results Nanoliposomal vaccine exposing PCSK9 peptide was found to provoke high-titers IgG antibody response against PCSK9 in rats, which was associated with the decrease plasma levels and function of plasma PCSK9. During the first week after STZ injection, it was indicated that the fasting blood glucose (FBG) level was 49% (−171.7±35 mg/dL, p Conclusions PCSK9 inhibition using liposomal vaccine can protect from glucose and insulin tolerance impairments in diabetic rats through an unknown and pancreatic-independent mechanism.

Background High oxidized phospholipid-apolipoprotein B-100 (OxPL-apoB) levels are associated with an increased risk for coronary heart disease (CHD). Genetic PCSK9 loss-of-function (LOF) variants result in life-long lower levels of LDL-C and lipoprotein(a) and reduced CHD risk, but the association with OxPL-apoB is unknown. Purpose To estimate the association between PCSK9 LOF variants and OxPL-apoB levels among black adults. Methods Genotyping for LOF variants (Y142X and C679X) was conducted for 10,196 black Reasons for Geographic And Racial Differences in Stroke study participants. OxPL-apoB was measured using antibody E06 for all participants with LOF variants (n=241) and randomly selected participants, matched at a 1:3 ratio, without LOF variants (n=723). Low OxPL-apoB was defined as the bottom quartile of the population distribution ( Results Adults with versus without PCSK9 LOF variants had lower LDL-C and lipoprotein(a) and were less likely to be taking a statin. (Table) A higher proportion of adults with versus without PCSK9 LOF variants had low OxPL-apoB levels (30.3 vs 23.4, p=0.03). After adjustment for covariates, the PR of low OxPL-apoB was increased for participants with compared to without LOF variants (PR 1.31, 95% CI 1.00, 1.72). Characteristics of REGARDS participants PCSK9 loss-of-function variant p-value Yes (n=241) No (n=723) Age, years, mean (SD) 63.7 (9.2) 63.8 (8.6) 0.81 Female, % 61.4 60.6 0.82 Diabetes, % 34.4 27.4 0.04 LDL-C, mg/dL, mean (SD) 85 (32) 118 (37) Conclusion Among black adults, PCSK9 LOF variants were associated with lower OxPL-apoB levels. Acknowledgement/Funding Industry/academic collaboration between Amgen Inc., University of Alabama at Birmingham and the Icahn School of Medicine at Mt. Sinai; and U01NS041588

Background Proprotein convertase subtilisin/kexin 9 (PCSK9) promotes hypercholesterolemia through reducing protein level of liver low-density lipoprotein (LDL) receptor (LDLR). Currently, PCSK9 inhibition is known as an efficient lipid-lowering approach. Purpose Here, we constructed a liposomal anti-PCSK9 vaccine, and evaluated its therapeutic effects on dyslipidemia and atherosclerosis in atherosclerotic mice. Methods Liposomal anti-PCSK9 vaccine was prepared viaattaching immunogenic peptide, termed Immunogenic Fused PCSK9-Tetanus (IFPT), on the surface of nanoliposome carriers by using DSPE-PEG-Maleimide lipid (L-IFPT). The L-IFPT formulation was adsorbed to Alum adjuvant (L-IFPTA+) and administrated subcutaneously four times with a bi-weekly interval in hypercholesterolemic C57BL/6 mice. Plasma levels of anti-PCSK9 antibody, plasma concentration of PCSK9 protein, effect of anti-PCSK9 antibody on PCSK9-LDLR interaction, protein levels of liver LDLR, lipid profile, as well as atherosclerotic lesion size and severity were measured in the vaccinated hypercholesterolemic mice. To determine immune safety, Inflammatory response was measured by evaluating IFN-γ and IL-10 producing splenic cells using ELISpot assay. Results L-IFPTA+vaccine promoted the high IgG antibody response against PCSK9 peptide in the hypercholesterolemic mice. L-IFPTA+-induced antibodies targeted plasma PCSK9 and thereby decreased plasma consecration of PCSK9, which was associated with the reduced PCSK9-LDLR interaction and the increased liver levels of LDLR protein. Inhibitory effect of L-IFPTA+vaccine was accompanied with a significant reduction of plasma levels of total cholesterol (TC), LDL-C, and VLDL-C. Interestingly, L-IFPTA+vaccine could considerably reduce atherosclerotic lesion size and intima to media thicknessin hypercholesterolemic mice. Long-term evaluation of hypercholesterolemic vaccinated mice showed that L-IFPTA+vaccine was able to promote a long-lasting anti-PCSK9 antibody titration, which was paralleled by a significant reduction of LDL-C over 16 weeks post prime immunization (Figure). Splenocytes isolated from vaccinated mice indicated the reduced IFN-γ producing cells and the elevated IL-10 producing cells, suggesting immune safety of liposomal anti-PCSK9 vaccine. Figure 1 Conclusions L-IFPTA+vaccine induced efficient anti-PCSK9 antibodies which could exert long-term therapeutic effect on hypercholesterolemia and atherosclerosis in mice, revealing a feasible vaccine-based approach for managing hypercholesterolemia and cardiovascular disease.

Background In patients with coronary artery disease (CAD), it remains unclear whether serum PCSK9 levels can predict the severity of the disease and the risk of future cardiovascular events. Methods Among the patients admitted for an acute myocardial infarction (MI) from September 2015 to December 2016 in an intensive care unit from a university hospital, serum PCSK9 levels were measured on admission in patients not previously receiving statin therapy. We aimed to evaluate the association between PCSK9 levels, metabolic parameters, severity of CAD on coronary angiography, and the risk of in-hospital events and at one-year follow-up. Results In a total of 648 patients (mean age: 66 years, 67% male), the median PCSK9 was 263 ng/ml, higher for females compared with males (270 vs 256 ng/ml, p=0.009). Serum PCSK9 was associated with LDL cholesterol (r=0.083, p=0.036), total cholesterol (r=0.136, p=0.001) and triglycerides (r=0.137, p=0.001). A positive association was also observed in the subgroup of patients with CRP >10 mg/L (p Conclusion In this large cohort of patients hospitalized for acute MI and not previously receiving statin therapy, PCSK9 levels was not associated with the severity or the recurrence of cardiovascular events. The clinical utility of measuring PCSK9 levels remains to be demonstrated for this category of patients.

Background Proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibition reduces plasma concentrations of most atherogenic lipoproteins such as low-density lipoprotein cholesterol (LDL-C), apolipoprotein B (apoB) and lipoprotein(a) [Lp(a)]. Atherogenic lipoprotein concentrations have also been linked with calcific aortic valve stenosis (CAVS). Purpose 1) To determine the association between genetic variants in PCSK9 and lipoprotein-lipid levels, 2) to determine whether loss of function (LOF) in PCSK9 is associated with CAVS and 3) to evaluate if PCSK9 could be implicated in aortic valve interstitial cells (VICs) calcification. Methods We built a weighted genetic risk score (wGRS) using 10 single nucleotide polymorphisms at the PCSK9 locus associated with LDL-C in the Global Lipids Genetics Consortium. We determined the association between the wGRS and LDL-C, apoB and Lp(a)] in 9692 participants of the EPIC-Norfolk study using linear regression. We investigated the association between the LOF PCSK9 R46L variant and CAVS risk in a meta-analysis of published (three Copenhagen studies, 1463 cases and 101,620 controls) and unpublished studies (UK Biobank, 1350 cases and 349,043 controls, Malmö Diet and Cancer study, 682 cases and 5963 controls and EPIC-Norfolk, 508 cases and 20,421 controls) prospective, population-based studies using logistic regression adjusted for age and sex. We evaluated PCSK9 expression and localization in explanted aortic valves by capillary Western blot and immunohistochemistry in patients with and without CAVS. Von Kossa staining was used to visualize aortic leaflet calcium deposits. We also assessed VICs calcification potential under oxidative stress condition. Results In EPIC-Norfolk, the wGRS was significantly associated with TC, LDL-C, and apoB (all p Conclusion PCSK9LOF variants are associated with lifelong reductions in non-Lp(a) apoB-containing lipoprotein levels and a lower risk of coronary artery disease and CAVS. PCSK9 is abundant in fibrotic and calcified aortic leaflets. Oxidative stress increases PCSK9 expression in VICs. These results support randomized clinical trials of PCSK9 inhibition in the prevention of CAVS.

Background Circulating proprotein convertase subtilisin/kexin type 9 (PCSK9) is a key regulator of LDL cholesterol levels and has emerged as a new therapeutic target in coronary artery disease (CAD). Plasma PCSK9 levels have also been related to other components of the lipid profile associated with atherosclerotic risk. Purpose Aim of the present study was to evaluate the relationship of plasma PCSK9 levels with lipid profile and measures of coronary atherosclerotic burden and risk in patients with stable CAD enrolled in the European EValuation of INtegrated Cardiac Imaging (EVINCI) study. Methods PCSK9 was measured in 412 patients (60.3±8.6 years, 256 males) with symptoms of stable CAD fully characterized by clinical risk factors, bio-humoral profiles, and treatment. All patients underwent coronary computed tomography (CT) angiography to assess the presence and characteristics of coronary atherosclerosis. We calculated an individual CT risk score, expressing the coronary atherosclerotic burden, combining extent, severity, composition, and location of plaques. Results Patients were divided in groups according to PCSK9 quartiles: I (266 ng/mL). LDL and HDL-cholesterol levels were significantly lower while total/HDL-cholesterol ratio was significantly higher in patients in the quartile I than in those in quartile IV (Figure A). CT angiography documented normal vessels in 30 and obstructive CAD in 35% of cases. Compared with patients with the highest levels (quartile IV), patients with the lowest PCSK9 levels (quartile I) had a higher CT risk score, a higher number of mixed plaque and higher hs-cTnI plasma levels (Figure B). PCSK9 itself was not associated with obstructive CAD. At multivariable analysis including clinical variables, medications and lipid variables PCSK9 was an independent predictor of the CT risk score (Coefficient - 0.129 (SE 0.03), p Figure 1 Conclusion PCSK9 levels are independently and inversely associated with the coronary atherosclerotic burden in patients with stable CAD. Acknowledgement/Funding AMGEN grant, EU FP7-CP-FP506 2007 project (grant agreement no. 222915)