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25-hydrocholesterol in macrophages increases inflammation and atherogenesis (ID 1390)
Cholesterol efflux pathways control T cell aging and atherosclerosis (ID 1391)
O026 - LINKING CELLULAR LIPID TRAFFICKING PROFILES OF INDIVIDUALS TO THE OUTCOMES OF CHOLESTEROL-LOWERING THERAPY IN THE GENERAL POPULATION (ID 311)
Abstract
Background and Aims
The outcomes of lipid-lowering therapy (LLT) vary between individuals. Previously we showed that for FH patients cellular lipid trafficking profiles are associated with on-treatment LDL-c levels (Pfisterer et al. CellRep Methods, doi:10.1016/j.crmeth.2022.100166). Here, we investigated how cellular profiles link with LDL-c levels, lipoprotein profiles, polygenic risk scores, and cardiovascular outcomes in the general population.
Methods
We used peripheral blood mononuclear cells (PBMCs) collected by THL Biobank as part of the Finnish population-based FINRISK 2012 study. We obtained samples for 400 subjects, including 200 recipients of LLT with available genetic, drug reimbursement, NMR metabolomic and longitudinal CVD outcome data. Using a multiplexed high-content imaging platform we obtained over 26 readouts for lipid uptake and storage in leukocytes for each sample.
Results
Cellular lipid uptake and storage were highly variable in our population-based study sample, and, in patients receiving either medium- or high-intensity statin monotherapy (n=39), were negatively correlated with serum LDL-cholesterol. Combining lipid uptake and storage readouts into cellular lipid trafficking scores (LTSs) improved this relationship (R=-0.45, p=0.0036). Subsequent combination of LTS with LDL-PRS produced even stronger correlation (R=-0.54, p=0.0004). Subjects in the lowest quintile of the LTS were at lower odds to be at their LDL target level (OR=0.068 CI [0.007,0.63], p=0.013) and at higher odds to experience MI or stroke (OR=30, 95% CI [2.64,339.75] p=0.004) in the 8-year follow-up period as compared to the rest of the group.
Conclusions
Cellular readouts provide novel insight into interindividual variation of LLT outcomes, providing new opportunities for treatment optimization and risk assessment for CVD prevention.
O027 - MYELOID-PCSK9 DEFICIENCY IMPROVES CARDIO-PROTECTION BY REGULATING LYVE1+ MACROPHAGES AGAINST ACUTE MYOCARDIAL INFARCTION (ID 492)
Abstract
Background and Aims
PCSK9 deficiency was reported to suppress inflammatory responses by regulating immune cells in the injury site. However, the effect of PCSK9-deficient macrophages after myocardial infarction (MI) has yet to be investigated. We hypothesized that PCSK9 deletion enabled cardio-protection during MI by regulating myeloid cells in the injury site. To prove this, we tested whether myeloid PCSK9 alters cardiac macrophage heterogeneity to adaptive remodeling after MI.
Methods
C57BL/6J, PCSK9-/-,PCSK9F/F, and Lyz2crePCSK9F/F male mice were subjected to MI using the left anterior descending branch of the coronary artery occlusion or sham operation. We analyzed PCSK9 deficiency functions in cardiac myeloid cells by Echocardiography, Trichrome staining, Flow cytometry, qPCR, and scRNA-seq.
Results
Myeloid-specific PCSK9 deletion alleviated cardiac dysfunction after myocardial injury. As a result of scRNA-seq, PCSK9 mediates macrophage heterogeneity following MI. Thus, the expression of CCR2-Lyve1+ cardiac macrophage subsets increased in PCSK9-/- MI hearts. These CCR2-Lyve1+ macrophages stimulate anti-inflammation against cardiac damage. Deficient PCSK9 in myeloid cells activates the BDNF pathway in the cardiac ischemic state to improve cell survival and wound healing compared to the control. These data elucidated that PCSK9 deletion affects macrophage heterogeneity to activate the BDNF signal, which protects against cardiac dysfunction.
Conclusions
PCSK9 deficiency in the myeloid activates CCR2-Lyve1+ cardiac macrophage to restrain the inflammatory state in the ischemic heart through the BDNF signaling pathway resulting in cardiac protection. Thus, myeloid PCSK9 can be a novel target to prevent adverse remodeling after MI.
O028 - INTRACELLULAR PROPROTEIN CONVERTASE SUBTILISIN/KEXIN TYPE 9 AND INFLAMMATION: UNVEILING THE ROLE OF EXTRACELLULAR VESICLES IN ATHEROMA FORMATION (ID 1071)
Abstract
Background and Aims
Extracellular vesicles (EVs), released by almost all cell types, are implicated in cell-to-cell communication. EVs play pro- and anti-atherothrombotic effects depending on their cargo, cell of origin, stimulus triggering the release. Vascular smooth muscle cells (VSMCs) can influence neighboring cells through bioactive molecules packed into EVs. VSMCs express and secrete proprotein convertase subtilisin/kexin type 9 (PCSK9), crucial for VSMCs differentiation, migration and proliferation. This study was aimed at unveiling the influence of intracellular PCSK9 on VSMCs-derived EVs.
Methods
EVs were isolated from VSMCs wild-type (VSMCWT-EVs) and VSMCs overexpressing PCSK9 (VSMCPCSK9-EVs). EVs were tested on endothelial cells, monocytes, macrophages and in Danio rerio zebrafish embryos. Techniques: flow cytometry, Western blot; nanoparticle tracking analysis; transmission electron microscopy; proteomic analysis; mitochondrial respiration; LDL uptake.
Results
VSMCPCSK9-EVs, compared to VSMCWT-EVs, carried a higher amount of PCSK9 and their miRNA content targeted 54 genes associated with atherosclerosis and inflammation. Concerning functional properties, VSMCPCSK9-EVs raised the expression of adhesion molecules in endothelial cells and that of pro-inflammatory cytokines in monocytes and macrophages. Migratory capacity of monocytes was increased with a switch toward a glycolytic phenotype. Secretome of monocytes exposed to VSMCPCSK9-EVs was enriched in pathways involved in immune response, immune effector processes, and cellular response to cytokines. Migratory capacity of macrophages was reduced along with a rise in the expression of CD36 and in the uptake of oxidized LDL. When injected in the hindbrain ventricle of zebrafish embryos, VSMCPCSK9-EVs favored a local recruitment of macrophages.
Conclusions
PCSK9 could play an inflammatory role by means of EVs released by VSMCs.
O029 - THE ANTI-INFLAMMATORY LNCRNA HEAT4 INTERACTS WITH THE PRO-INFLAMMATORY PROTEIN IP1 IN NON-CLASSICAL MONOCYTES PROMOTING VASCULAR REGENERATION (ID 775)
Abstract
Background and Aims
Using next-generation-sequencing, we identified the lncRNA Heat4 which is significantly upregulated in patients with ischemic cardiomyopathy compared to controls. Here, we aim to characterize Heat4's function and identify potential interaction-partners.
Methods
We performed poly-A-fractionation, qPCR and single-cell-sequencing to study Heat4’s function in vitro and used a carotid-injury model for in vivo-studies. Pulldown of Heat4 with subsequent mass spectrometry was used to identify potential Heat4-interaction-partners.
Results
Heat4 is located in the cytoplasm of non-classical monocytes where it is stabilized by its poly-A-tail (+5.30-fold-enrichment in poly(A+)-fraction; p<0.05). Matching the known anti-inflammatory properties of non-classical monocytes, Heat4 mediates anti-inflammatory functions in vitro (Heat4-overexpression: -38.6% TNFα-RNA-reduction; p<0.05) and promotes vascular regeneration in vivo (carotid arteries of NOD-SCID mice regenerated faster after injection of human monocytes with Heat4-overexpression compared to control-monocytes (% regenerated area: +1.85-fold-enrichment; N=6; p<0.05)). In addition, an increased expression of the non-classical monocyte marker CD16 was found in monocytes after Heat4-overexpression (+2.37-fold-enrichment; p<0.05). We identified IP1 and IP2 as Heat4-interaction-partners (+1.20 and +1.45-fold-enrichment in Heat4-fraction compared to control-probe). While IP1 was validated as Heat4-interaction-partner, IP2 was probably only detected as part of the known heterodimer with IP1. Lower IP1- than IP1/IP2-concentrations were found in both monocyte cell lysate and supernatant. Heat4-overexpression resulted in reduced extracellular levels of the IP1/IP2-heterodimer (IP1/IP2: -23.6%; p<0.05) but not IP1 alone.
Conclusions
The lncRNA Heat4 is elevated in the blood of patients with heart failure and limits the inflammatory response of non-classical monocytes. Modulating Heat4 levels may represent a novel strategy for treatment of cardiovascular diseases with impaired vascular functions.
O030 - DUAL ELEVATED REMNANT CHOLESTEROL AND C-REACTIVE PROTEIN IN MYOCARDIAL INFARCTION, ATHEROSCLEROTIC CARDIOVASCULAR DISEASE, AND MORTALITY. (ID 807)
Abstract
Background and Aims
Elevated remnant cholesterol and low-grade inflammation each cause atherosclerotic cardiovascular disease (ASCVD); however, it is unknown whether joint elevation of both factors confers the highest risk. We tested the hypothesis that dual elevated remnant cholesterol and low-grade inflammation marked by elevated C-reactive protein is associated with the highest risk of myocardial infarction, ASCVD, and all-cause mortality.
Methods
The Copenhagen General Population Study randomly recruited white Danish individuals aged 20-100 years in 2003-2015 and followed them for a median 9.5 years. ASCVD was cardiovascular mortality, myocardial infarction, stroke, and coronary revascularization.
Results
In 103,221 individuals, we observed 2,454 (2.4%) myocardial infarctions, 5,437 (5.3%) ASCVD events, and 10,521 (10.2%) deaths. The hazard ratios increased with each of stepwise higher remnant cholesterol and stepwise higher C-reactive protein. In individuals with the highest tertile of both remnant cholesterol and C-reactive protein compared to individuals with the lowest tertile of both, the multivariable adjusted hazard ratios were 2.2(95%CI:1.9-2.7) for myocardial infarction, 1.9(1.7-2.2) for ASCVD, and 1.4(1.3-1.5) for all-cause mortality. Corresponding values for only the highest tertile of remnant cholesterol were 1.4(1.2-1.8), 1.2(1.0-1.4), and 1.1(1.0-1.2), and those for only the highest tertile of C-reactive protein were 1.6(1.3-2.0), 1.5(1.3-1.7), and 1.3(1.2-1.5), respectively. There was no statistical evidence for interaction between elevated remnant cholesterol and elevated C-reactive protein on risk of myocardial infarction (P=0.10), ASCVD (P=0.40), or all-cause mortality (P=0.74).
Conclusions
Dual elevated remnant cholesterol and C-reactive protein confers the highest risk of myocardial infarction, ASCVD, and all-cause mortality.