Inhibition of PFKFB3 Hampers the Progression of Atherosclerosis and Promotes Plaque Stability

Aims: 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB)3-mediated glycolysis is pivotal in driving macrophage- and endothelial cell activation and thereby inflammation. Once activated, these cells play a crucial role in the progression of atherosclerosis. Here, we analyzed the expression of PFKFB3 in human atherosclerotic lesions and investigated the therapeutic potential of pharmacological inhibition of PFKFB3 in experimental atherosclerosis by using the glycolytic inhibitor PFK158. Methods and Results: PFKFB3 expression was higher in vulnerable human atheromatous carotid plaques when compared to stable fibrous plaques and predominantly expressed in plaque macrophages and endothelial cells. Analysis of advanced plaques of human coronary arteries revealed a positive correlation of PFKFB3 expression with necrotic core area. To further investigate the role of PFKFB3 in atherosclerotic disease progression, we treated 6–8 weeks old male Ldlr–/– mice. These mice were fed a high cholesterol diet for 13 weeks, of which they were treated for 5 weeks with the glycolytic inhibitor PFK158 to block PFKFB3 activity. The incidence of fibrous cap atheroma (advanced plaques) was reduced in PFK158-treated mice. Plaque phenotype altered markedly as both necrotic core area and intraplaque apoptosis decreased. This coincided with thickening of the fibrous cap and increased plaque stability after PFK158 treatment. Concomitantly, we observed a decrease in glycolysis in peripheral blood mononuclear cells compared to the untreated group, which alludes that changes in the intracellular metabolism of monocyte and macrophages is advantageous for plaque stabilization. Conclusion: High PFKFB3 expression is associated with vulnerable atheromatous human carotid and coronary plaques. In mice, high PFKFB3 expression is also associated with a vulnerable plaque phenotype, whereas inhibition of PFKFB3 activity leads to plaque stabilization. This data implies that inhibition of inducible glycolysis may reduce inflammation, which has the ability to subsequently attenuate atherogenesis

Nile Red Quantifier:A novel and quantitative tool to study lipid accumulation in patient-derived circulating monocytes using confocal microscopy

The inflammatory profile of circulating monocytes is an important biomarker for atherosclerotic plaque vulnerability. Recent research revealed that peripheral lipid uptake by monocytes alters their phenotype toward an inflammatory state and this coincides with an increased lipid droplet (LD) content. Determination of lipid content of circulating monocytes is, however, not very well established. Based on Nile Red (NR) neutral LD imaging, using confocal microscopy and computational analysis, we developed NR Quantifier (NRQ), a novel quantification method to assess LD content in monocytes. Circulating monocytes were isolated from blood and used for the NR staining procedure. In monocytes stained with NR, we clearly distinguished, based on 3D imaging, phospholipids and exclusively intracellular neutral lipids. Next, we developed and validated NRQ, a semi-automated quantification program that detects alterations in lipid accumulation. NRQ was able to detect LD alterations after ex vivo exposure of isolated monocytes to freshly isolated LDL in a time-and dose-dependent fashion. Finally, we validated NRQ in patients with familial hypercholesterolemia and obese subjects in pre- and postprandial state. In conclusion, NRQ is a suitable tool to detect even small differences in neutral LD content in circulating monocytes using NR staining

Atherogenic Lipoprotein(a) Increases Vascular Glycolysis, Thereby Facilitating Inflammation and Leukocyte Extravasation

Rationale: Patients with elevated levels of lipoprotein(a) [Lp(a)] are hallmarked by increased metabolic activity in the arterial wall on positron emission tomography/computed tomography, indicative of a proinflammatory state. Objective: We hypothesized that Lp(a) induces endothelial cell inflammation by rewiring endothelial metabolism. Methods and Results: We evaluated the impact of Lp(a) on the endothelium and describe that Lp(a), through its oxidized phospholipid content, activates arterial endothelial cells, facilitating increased transendothelial migration of monocytes. Transcriptome analysis of Lp(a)-stimulated human arterial endothelial cells revealed upregulation of inflammatory pathways comprising monocyte adhesion and migration, coinciding with increased 6-phophofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB)-3-mediated glycolysis. ICAM (intercellular adhesion molecule)-1 and PFKFB3 were also found to be upregulated in carotid plaques of patients with elevated levels of Lp(a). Inhibition of PFKFB3 abolished the inflammatory signature with concomitant attenuation of transendothelial migration. Conclusions: Collectively, our findings show that Lp(a) activates the endothelium by enhancing PFKFB3-mediated glycolysis, leading to a proadhesive state, which can be reversed by inhibition of glycolysis. These findings pave the way for therapeutic agents targeting metabolism aimed at reducing inflammation in patients with cardiovascular disease

Discovery and systematic characterization of risk variants and genes for coronary artery disease in over a million participants

The discovery of genetic loci associated with complex diseases has outpaced the elucidation of mechanisms of disease pathogenesis. Here we conducted a genome-wide association study (GWAS) for coronary artery disease (CAD) comprising 181,522 cases among 1,165,690 participants of predominantly European ancestry. We detected 241 associations, including 30 new loci. Cross-ancestry meta-analysis with a Japanese GWAS yielded 38 additional new loci. We prioritized likely causal variants using functionally informed fine-mapping, yielding 42 associations with less than five variants in the 95% credible set. Similarity-based clustering suggested roles for early developmental processes, cell cycle signaling and vascular cell migration and proliferation in the pathogenesis of CAD. We prioritized 220 candidate causal genes, combining eight complementary approaches, including 123 supported by three or more approaches. Using CRISPR-Cas9, we experimentally validated the effect of an enhancer in MYO9B, which appears to mediate CAD risk by regulating vascular cell motility. Our analysis identifies and systematically characterizes >250 risk loci for CAD to inform experimental interrogation of putative causal mechanisms for CAD. 2022, The Author(s).T. Kessler is supported by the Corona-Foundation (Junior Research Group Translational Cardiovascular Genomics) and the German Research Foundation (DFG) as part of the Sonderforschungsbereich SFB 1123 (B02). T.J. was supported by a Medical Research Council DTP studentship (MR/S502443/1). J.D. is a British Heart Foundation Professor, European Research Council Senior Investigator, and National Institute for Health and Care Research (NIHR) Senior Investigator. J.C.H. acknowledges personal funding from the British Heart Foundation (FS/14/55/30806) and is a member of the Oxford BHF Centre of Research Excellence (RE/13/1/30181). R.C. has received funding from the British Heart Foundation and British Heart Foundation Centre of Research Excellence. O.G. has received funding from the British Heart Foundation (BHF) (FS/14/66/3129). P.S.d.V. was supported by American Heart Association grant number 18CDA34110116 and National Heart, Lung, and Blood Institute grant R01HL146860. The Atherosclerosis Risk in Communities study has been funded in whole or in part with Federal funds from the National Heart, Lung and Blood Institute, National Institutes of Health, Department of Health and Human Services (contract HHSN268201700001I, HHSN268201700002I, HHSN268201700003I, HHSN268201700004I and HHSN268201700005I), R01HL087641, R01HL059367 and R01HL086694; National Human Genome Research Institute contract U01HG004402; and National Institutes of Health contract HHSN268200625226C. We thank the staff and participants of the ARIC study for their important contributions. Infrastructure was partly supported by grant UL1RR025005, a component of the National Institutes of Health and NIH Roadmap for Medical Research. The Trøndelag Health Study (The HUNT Study) is a collaboration between HUNT Research Centre (Faculty of Medicine and Health Sciences, NTNU, Norwegian University of Science and Technology), Trøndelag County Council, Central Norway Regional Health Authority and the Norwegian Institute of Public Health. The K.G. Jebsen Center for Genetic Epidemiology is financed by Stiftelsen Kristian Gerhard Jebsen; Faculty of Medicine and Health Sciences, NTNU, Norwegian University of Science and Technology; and Central Norway Regional Health Authority. Whole genome sequencing for the HUNT study was funded by HL109946. The GerMIFs gratefully acknowledge the support of the Bavarian State Ministry of Health and Care, furthermore founded this work within its framework of DigiMed Bayern (grant DMB-1805-0001), the German Federal Ministry of Education and Research (BMBF) within the framework of ERA-NET on Cardiovascular Disease (Druggable-MI-genes, 01KL1802), within the scheme of target validation (BlockCAD, 16GW0198K), within the framework of the e:Med research and funding concept (AbCD-Net, 01ZX1706C), the British Heart Foundation (BHF)/German Centre of Cardiovascular Research (DZHK)-collaboration (VIAgenomics) and the German Research Foundation (DFG) as part of the Sonderforschungsbereich SFB 1123 (B02), the Sonderforschungsbereich SFB TRR 267 (B05), and EXC2167 (PMI). This work was supported by the British Heart Foundation (BHF) under grant RG/14/5/30893 (P.D.) and forms part of the research themes contributing to the translational research portfolios of the Barts Biomedical Research Centre funded by the UK National Institute for Health Research (NIHR). I.S. is supported by a Precision Health Scholars Award from the University of Michigan Medical School. This work was supported by the European Commission (HEALTH-F2–2013-601456) and the TriPartite Immunometabolism Consortium (TrIC)-NovoNordisk Foundation (NNF15CC0018486), VIAgenomics (SP/19/2/344612), the British Heart Foundation, a Wellcome Trust core award (203141/Z/16/Z to M.F. and H.W.) and the NIHR Oxford Biomedical Research Centre. M.F. and H.W. are members of the Oxford BHF Centre of Research Excellence (RE/13/1/30181). The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health. C.P.N. and T.R.W. received funding from the British Heart Foundation (SP/16/4/32697). C.J.W. is funded by NIH grant R35-HL135824. B.N.W. is supported by the National Science Foundation Graduate Research Program (DGE, 1256260). This research was supported by BHF (SP/13/2/30111) and conducted using the UK Biobank Resource (application 9922). O.M. was funded by the Swedish Heart and Lung Foundation, the Swedish Research Council, the European Research Council ERC-AdG-2019-885003 and Lund University Infrastructure grant ‘Malmö population-based cohorts’ (STYR 2019/2046). T.R.W. is funded by the British Heart Foundation. I.K., S. Koyama, and K. Ito are funded by the Japan Agency for Medical Research and Development, AMED, under grants JP16ek0109070h0003, JP18kk0205008h0003, JP18kk0205001s0703, JP20km0405209 and JP20ek0109487. The Biobank Japan is supported by AMED under grant JP20km0605001. J.L.M.B. acknowledges research support from NIH R01HL125863, American Heart Association (A14SFRN20840000), the Swedish Research Council (2018-02529) and Heart Lung Foundation (20170265) and the Foundation Leducq (PlaqueOmics: New Roles of Smooth Muscle and Other Matrix Producing Cells in Atherosclerotic Plaque Stability and Rupture, 18CVD02. A.V.K. has been funded by grant 1K08HG010155 from the National Human Genome Research Institute. K.G.A. has received support from the American Heart Association Institute for Precision Cardiovascular Medicine (17IFUNP3384001), a KL2/Catalyst Medical Research Investigator Training (CMeRIT) award from the Harvard Catalyst (KL2 TR002542) and the NIH (1K08HL153937). A.S.B. has been supported by funding from the National Health and Medical Research Council (NHMRC) of Australia (APP2002375). D.S.A. has received support from a training grant from the NIH (T32HL007604). N.P.B., M.C.C., J.F. and D.-K.J. have been funded by the National Institute of Diabetes and Digestive and Kidney Diseases (2UM1DK105554). EPIC-CVD was funded by the European Research Council (268834) and the European Commission Framework Programme 7 (HEALTH-F2-2012-279233). The coordinating center was supported by core funding from the UK Medical Research Council (G0800270; MR/L003120/1), British Heart Foundation (SP/09/002, RG/13/13/30194, RG/18/13/33946) and NIHR Cambridge Biomedical Research Centre (BRC-1215-20014). The views expressed are those of the author(s) and not necessarily those of the NIHR or the Department of Health and Social Care. This work was supported by Health Data Research UK, which is funded by the UK Medical Research Council, Engineering and Physical Sciences Research Council, Economic and Social Research Council, Department of Health and Social Care (England), Chief Scientist Office of the Scottish Government Health and Social Care Directorates, Health and Social Care Research and Development Division (Welsh Government), Public Health Agency (Northern Ireland), British Heart Foundation and Wellcome. Support for title page creation and format was provided by AuthorArranger, a tool developed at the National Cancer Institute.Scopu

Omecamtiv mecarbil in chronic heart failure with reduced ejection fraction, GALACTIC‐HF: baseline characteristics and comparison with contemporary clinical trials

Aims: The safety and efficacy of the novel selective cardiac myosin activator, omecamtiv mecarbil, in patients with heart failure with reduced ejection fraction (HFrEF) is tested in the Global Approach to Lowering Adverse Cardiac outcomes Through Improving Contractility in Heart Failure (GALACTIC‐HF) trial. Here we describe the baseline characteristics of participants in GALACTIC‐HF and how these compare with other contemporary trials. Methods and Results: Adults with established HFrEF, New York Heart Association functional class (NYHA) ≥ II, EF ≤35%, elevated natriuretic peptides and either current hospitalization for HF or history of hospitalization/ emergency department visit for HF within a year were randomized to either placebo or omecamtiv mecarbil (pharmacokinetic‐guided dosing: 25, 37.5 or 50 mg bid). 8256 patients [male (79%), non‐white (22%), mean age 65 years] were enrolled with a mean EF 27%, ischemic etiology in 54%, NYHA II 53% and III/IV 47%, and median NT‐proBNP 1971 pg/mL. HF therapies at baseline were among the most effectively employed in contemporary HF trials. GALACTIC‐HF randomized patients representative of recent HF registries and trials with substantial numbers of patients also having characteristics understudied in previous trials including more from North America (n = 1386), enrolled as inpatients (n = 2084), systolic blood pressure < 100 mmHg (n = 1127), estimated glomerular filtration rate < 30 mL/min/1.73 m2 (n = 528), and treated with sacubitril‐valsartan at baseline (n = 1594). Conclusions: GALACTIC‐HF enrolled a well‐treated, high‐risk population from both inpatient and outpatient settings, which will provide a definitive evaluation of the efficacy and safety of this novel therapy, as well as informing its potential future implementation

Metabolism: The road to inflammation and atherosclerosis

PURPOSE OF REVIEW: Evidence accumulates suggesting that cellular metabolic alterations fuel and dictate the inflammatory state of cells. In this review, we provide an overview of the observed metabolic reprogramming in endothelial cells and innate immune cells upon interaction with modified lipoproteins, thereby contributing to the progression of atherosclerosis

The role of (Modified) lipoproteins in vascular function: A duet between monocytes and the endothelium

Over the last century, many studies have demonstrated that low-density lipoprotein (LDL) is a key risk factor of cardiovascular diseases (CVD) related to atherosclerosis. Thus, for these CVD patients, LDL lowering agents are commonly used in the clinic to reduce the risk for CVD. LDL, upon modification, will develop distinct inflammatory and proatherogenic potential, leading to impaired endothelial integrity, influx of immune cells and subsequent increased foam cell formation. LDL can also directly affect peripheral monocyte composition, rendering them in a more favorable position to migrate and accumulate in the subendothelial space. It has become apparent that other lipoprotein particles, such as triglyceride-rich lipoproteins or remnants (TRL) and lipoprotein(a) [Lp(a)] may also impact on atherogenic pathways. Evidence is accumulating that Lp(a) can promote peripheral monocyte activation, eventually leading to increased transmigration through the endothelium. Similarly, remnant cholesterol has been identified to play a key role in endothelial dysfunction and monocyte behavior. In this review, we will discuss recent developments in understanding the role of different lipoproteins in the context of inflammation at both the level of the monocyte and the endothelium

Lipoprotein(A) as orchestrator of calcific aortic valve stenosis

Aortic valve stenosis (AVS) is the most prevalent disease in the Western World with exponentially increased incidence with age. If left untreated, the yearly mortality rates increase up to 25%. Currently, no effective pharmacological interventions have been established to treat or prevent AVS. The only treatment modality so far is surgical or transcatheter aortic valve replacement (AVR). Lipoprotein(a) [Lp(a)] has been implicated as a pivotal player in the pathophysiology of calcification of the valves. Patients with elevated levels of Lp(a) have a higher risk of hospitalization or mortality due to the presence of AVS. Multiple studies indicated Lp(a) as a likely causal and independent risk factor for AVS. This review discusses the most important findings and mechanisms related to Lp(a) and AVS in detail. During the progression of AVS, Lp(a) enters the aortic valve tissue at damaged sites of the valves. Subsequently, autotaxin converts lysophosphatidylcholine in lysophosphatidic acid (LysoPA) which in turn acts as a ligand for the LysoPA receptor. This triggers a nuclear factor-κB cascade leading to increased transcripts of interleukin 6, bone morphogenetic protein 2, and runt-related transcription factor 2. This progresses to the actual calcification of the valves through production of alkaline phosphatase and calcium depositions. Furthermore, this review briefly mentions potentially interesting therapies that may play a role in the treatment or prevention of AVS in the near future

The maturation of a "neural-hematopoietic' inflammatory axis in cardiovascular disease

Purpose of reviewAtherogenesis is the result of a complex interplay between lipids and innate immune cells, which are descendants of upstream progenitors residing in hematopoietic organs. In this review, we will discuss recent advances in the connection between hematopoiesis and atherogenesis.Recent findingsThe relevance of a neural-hematopoietic axis was recently supported by the demonstration of a correlation between metabolic activity in the amygdala and the bone marrow. During follow-up, both amygdalar and bone marrow activities also predicted cardiovascular risk in patients, lending further support to a connection between neural stress and cardiovascular events mediated via increased hematopoietic activity.In parallel, functional changes in hematopoietic stem cells may also convey cardiovascular risk. In experimental models, knock-out of the ten-eleven translocation 2 (TET2) gene leading to monocyte-macrophage hyperresponsiveness, was associated with accelerated atherogenesis in murine experiments. In humans, whole-exome sequencing reporting on the clonal hematopoiesis of indeterminate potential' gene substantiated a two-fold elevated risk for developing coronary heart disease compared with noncarriers.SummaryRecent studies support the relevance of a neural-hematopoietic' inflammatory axis and clonal hematopoiesis as drivers of atherogenesis in humans. These data warrant further studies addressing the role of novel hematopoietic' targets for the treatment of patients with increased cardiovascular ris