Conference Agenda

Most atherosclerosis-prone blood vessels are surrounded by perivascular adipose tissue (PVAT), which is contiguous with the adventitial layer of arteries. PVAT is a physiologically and metabolically active endocrine tissue that regulates vascular biology through paracrine signalling. Adipose tissue macrophages (ATMs) within PVAT are key players in obesity-associated inflammation, metabolic stress and atherosclerosis due to their capacity to switch from an alternatively activated anti-inflammatory M2 to a classically activated pro-inflammatory M1 phenotype. The receptor ChemR23 appears to play a crucial role in regulating this phenotypic switching and thereby influences the crosstalk between PVAT and arteries during atherosclerosis.

We use an atherosclerotic mouse model with a systemic knockout of ChemR23 expressing eGFP (Apoe^-/-ChemR23-knockout/knockin mice). These mice are fed either a Normal Chow Diet (NCD) or a Western Diet (WD) for 4 weeks. White and brown adipose tissue depots, particularly PVAT, are analysed by flow cytometry and immunohistochemistry. In addition, macrophages are metabolically characterized using SCENITH, and their phenotype is determined by immunofluorescence and flow cytometry.

After 4 weeks of WD, the weight of adipose tissue depots was increased, accompanied by adipocyte hypertrophy and whitening of brown adipose tissue (BAT). The number of macrophages was elevated across all adipose tissues, indicating an inflammatory state. This was associated with reduced PDGFRα⁺ adipose progenitor cells and lower plasma levels of IL-33 and FGF21, both key effectors of adipose tissue beiging. Interestingly, bone marrow-derived macrophages (BMDMs) from ChemR23 knockout mice exhibited a metabolic M2-like phenotype. FACS-sorted PVAT macrophages (CD45⁺CD11b⁺) are currently analysed at the transcriptomic level using single-cell RNA sequencing.

Future studies will investigate PVAT immune cell types and their immunometabolic influence on the aorta under homeostatic and inflammatory conditions using spatial biology (MACSima™ Imaging Cyclic Staining, MICS) and adipocyte–macrophage cell culture assays to elucidate ChemR23-dependent mechanisms of cellular crosstalk.

Introduction: The present study aimed to enhance the endogenous expression of apolipoprotein A1 (apoA1) or paraoxonase 1 (PON1) in the liver of apolipoprotein E deficient (apoE^-/-) mice and to investigate their atheroprotective effects. ApoA1, the major structural component of HDL, facilitates reverse cholesterol transport, while PON1, an HDL-associated enzyme, exerts potent antioxidant effects. Together, these molecules contribute to the atheroprotective activity of HDL, but efficient therapies to increase their levels in pathological conditions are not yet available. The clustered regularly interspaced short palindromic repeats (CRISPR)/deactivated CRISPR-associated protein 9 (dCas9) enables the control of gene expression without DNA editing, and has generated interest for therapeutic applications due to its high efficiency and specificity.

Methods: CRISPR/dCas9 plasmids were used to transcriptionally activate the endogenous ApoA1/PON1 genes in apoE^−/− mice, which is a common animal model of spontaneous atherosclerosis. The ApoA1/PON1 and control plasmids (40 µg DNA plus in vivo-jetPEI^®) were administered by tail vein injection and the animals were sacrificed 30 days after that.

Results: The results showed that the endogenous ApoA1/PON1 genes in the liver of apoE^−/− mice were successfully overexpressed by using CRISPR/dCas9 system, as it was proven by the significantly increased gene and protein expression of these molecules in liver tissue homogenates. Most importantly, ApoA1 and PON1 stimulation was maintained for four weeks after a single dose of CRISPR/dCas9 plasmids. Results also showed an increase in the levels of ApoaA1 and PON1 in the sera of mice. Notably, the increased serum apoA1 was distributed between HDL (83%), low density lipoproteins (LDL) (200%) and its lipid-free form (140%) compared to the corresponding lipoprotein fractions isolated from the Control group. We have demonstrated that the overexpression of ApoA1 in the liver led to the increase of cholesterol levels in serum HDL, liver and gallbladder. The implicated mechanisms involved the upregulation of hepatic scavenger receptor class B1 (SCARB1), 7α-hydroxylase (CYP7A1), and ATP-binding cassette sub-family G member 8 (ABCG8) transporter. The stimulation of PON1 led to an increase in the antioxidant potential of the serum (measured as Thiobarbituric Acid Reactive Substances - TBARS and PON1 enzymatic activity). A very important finding of our study was that the area of the lipid deposits (stained by Oil Red O) in the thoracic aorta was markedly reduced in the treated mice.

Conclusions: We successfully activated the endogenous hepatic apoA-I and PON1 genes by using the CRISPR/dCas9 system in apoE^−/− mice and we demonstrated that the liver-secreted apoA-I or PON1 impede atheroma progression. This approach initiates the way for the future use of CRISPR/dCas9 transcriptional activation of endogenous genes for atherosclerosis treatment.

Funding: This research was funded by the Romanian Academy and by the PNRR program, CF 197-2022/PNRR-III-C9-2022-I8 (760059/23.05.2023).

Background – Obesity and diabetes are important cardiovascular risk factors that are accelerate biological aging. On microscopical level, aging is defined as cellular senescence, characterized by permanent cell cycle inhibition, disturbed cell-cell communication and resistance to apoptosis. Accumulation of cellular senescence contributes to increased susceptibility to ventricular arrhythmias (VA). The application of senolytic compounds, that selectively eliminate senescent cells, may offer a novel therapeutic approach to reduce VAs in aging and metabolically-challenged hearts.

Methods and Results – In a clinical cohort, patients were stratified by body mass index above or below 30 to assess VA inducibility. Patients with obesity (BMI > 30) displayed increased VA vulnerability. To study the mechanisms underlying the relation between obesity and arrythmia, male C57BL/6J mice were fed a high-fat diet for 12 weeks to induce diet-induced obesity (DIO). Animals were treated with vehicle or a senolytic combination of dasatinib (5 mg/kg) and quercetin (50 mg/kg), administered orally three times within one week. Cardiac senescence was assessed by quantifying Cdkn1a and Cdkn2a mRNA expression using qPCR and was further confirmed by senescence-associated β-galactosidase staining. Senolytics-treated DIO mice showed a significant reduction in cardiac senescence compared to controls. Senolytic treatment reduced susceptibility to VAs, as demonstrated by programmed ventricular stimulation and epicardial fluorescence imaging in isolated perfused hearts. Cardiac fibroblasts were identified as the predominant senescent population and the principal source of arrhythmia-stimulating IL-6 by single cell RNA sequencing and flow cytometry.

Histological analysis and qPCR of fibrosis-associated genes showed attenuated myocardial fibrosis following senolysis, consistent with the susceptibility of cardiac fibroblasts to senolytic treatment. Furthermore, senolytic treatment improved mitochondrial respiration and network organization. In vitro, conditioned medium from senescent human cardiac fibroblasts impaired mitochondrial network complexity in tdTomato-COX8–labeled human cardiomyocytes, an effect abrogated by IL-6 neutralization.

Conclusion – Senescent fibroblasts contribute to obesity- and metabolically-associated cardiovascular dysfunction by promoting IL-6 production and cardiac fibrosis. Administration of senolytics reduces cardiac senescence, fibrosis, and decreases the susceptibility of VAs in preclinical models. These findings underscore the therapeutic potential of targeting senescent fibroblasts to prevent arrhythmias in obesity- and metabolism-related cardiovascular disorders.

Background: Atherosclerosis is a lipid-driven vascular inflammation in which macrophages play a central role in plaque initiation and progression. Excessive cholesterol uptake drives metabolic dysregulation in macrophages, promoting foam cell formation and disease progression. The mitochondrial TCA cycle enzyme fumarate hydratase (FH1) has been implicated in macrophage metabolic regulation and pro-atherosclerotic cytokine release, but its role in foam cell formation and atherosclerosis remains entirely unknown.

Aim: To investigate the role of FH1 in foam cell formation and atherosclerotic plaque progression, and to explore its potential clinical relevance in humans.

Methods/Results: In silico analysis of single-cell RNA sequencing of leukocytes from murine atherosclerotic aortas revealed decreased Fh1 expression in Trem2⁺ macrophages. Immunofluorescent imaging of atherosclerotic plaques in low-density lipoprotein receptor knock-out (LDLr^-/-) mice fed western-type diet for 12 weeks confirmed reduced Fh1 abundance in CD68^pos BODIPY^pos foamy macrophages compared to CD68^pos BODIPY^neg macrophages (n=3; P<0.001). FACS confirmed decreased FH1 abundance in aortic foamy macrophages in LDLr-/- mice (n=4/group; P=0.029). In silico CRISPR screen-analysis of BV2 myeloid cells undergoing foam cell formation ranked Fh1 as the TCA enzyme most associated with oxLDL uptake (n=5/group; P<0.001). Interestingly, in vitro, Fh1 expression was unchanged in oxLDL-treated bone marrow-derived macrophages (BMDMs), but co-stimulation with LPS, mimicking plaque inflammation, significantly reduced FH1 (n=3/group; P=0.001). Compared to vehicle, pre-stimulation with the FH1 inhibitor (FHIN1) reduced DIL-labeled oxLDL uptake in BMDMs (n=3/group; P=0.015). Genetic Fh1-deficiency in BMDMs reduced expression of foam cell associated genes including Trem2 (n=3/group; P=0.001), Cd36 (P=0.034) and Plin2 (P=0.044). In vivo, macrophage-specific Fh1 deletion (Cx3cr1-CreERT2 x Fh1-flox-flox (Cx3cr1^Fh1) mice) showed reduced Fh1 expression in peritoneal Cx3cr1⁺ leukocytes after 1 week of tamoxifen-containing HFD ((TAM-HFD) n =3/group; P=0.034). In comparison to controls, transplantation of Cx3cr1^Fh1 bone marrow into lethally-irradiated LDLr^-/- mice increased atherosclerotic burden after 12 weeks of HFD followed by 4 weeks TAM-HFD (n=7/group; P=0.021). Ongoing work is investigating phenotypic and inflammatory changes in the plaque microenvironment. In 1071 patients undergoing carotid endarterectomy, higher FH1 expression in plaques was associated with lower occurrence of stroke/TIA at the time of surgery (OR 0.84; 95% CI 0.73–0.96; P=0.009), independent of risk factors and lipid biomarkers.

Conclusion: Fh1 is downregulated in foam cells both in vitro and in vivo, and higher Fh1 expression in human plaques is associated with reduced cerebrovascular events, suggesting potential predictive relevance. FH1 deficiency decreased oxLDL uptake, altered foam cell gene expression, and increased atherosclerotic burden in mice. These findings highlight FH1 as a potential target for atherosclerosis prevention and therapy.

Background: Systemic low-grade inflammation is a key driver of atherosclerotic cardiovascular disease (ASCVD). While high-sensitivity C-reactive protein (hsCRP) is the guideline-recommended biomarker reflecting residual inflammatory risk, the clinical utility of leukocyte-derived scores, such as the neutrophil-to-lymphocyte ratio (NLR) and further indices, remains unclear in hospitalized patients with ASCVD. More information from large, well-characterized cardiovascular (CV) cohorts comparing hsCRP with leukocyte-derived inflammatory indices using contemporary approaches for head-to-head comparison will help clarify their combined clinical utility for risk assessment in high-risk patients.

Methods: In this prospective, single-center cohort study, inflammatory biomarkers including hsCRP, differential leukocyte counts, and leukocyte-derived scores were analyzed in 1649 hospitalized patients. The primary endpoint of this analysis was a composite of non-fatal myocardial infarction, non-fatal stroke and CV death (MACE: major adverse cardiovascular events). Kaplan-Meier and uni- and multivariable Cox regression analyses were performed. Model performance of biomarkers was compared with likelihood ratio Chi² statistics to assess incremental value for prediction of MACE.

Results: During a median follow-up period of two years, MACE was observed in 114 out of 1649 subjects. Kaplan-Meier curve analyses demonstrated higher incidence of MACE in individuals with hsCRP levels above the guideline-based cut-off of 2 mg/L or those with an NLR above the cohort median of 2.28 (log-rank p (hsCRP) = 0.031; log-rank p (NLR) <0.001). Combined assessment of hsCRP and NLR identified those patients with both markers elevated at highest risk, while risk was intermediate when only one marker was elevated and lowest when both were below the cut-off (log-rank p=0.002). These findings were confirmed in Cox regression analyses showing a 2.8-fold higher risk of MACE in those with high NLR and hsCRP compared with those with low levels of both (Hazard ratio (HR): 2.76; 95% confidence interval (CI): 1.57, 4.84; p<0.001). In further univariable analyses, hsCRP, neutrophil percentage, lymphocyte count and percentage, NLR, neutrophil-to-monocyte ratio (NMR), lymphocyte-to-monocyte ratio (LMR) and monocyte-to-lymphocyte ratio (MLR) were associated with MACE. In multivariable analyses adjusted for age, sex, body mass index, systolic blood pressure, type 2 diabetes, smoking, creatinine, and low-density lipoprotein cholesterol, only hsCRP, neutrophil percentage, lymphocyte percentage, NLR and NMR, but not LMR and MLR, remained independently associated with MACE. In the extended models, hsCRP provided the highest incremental model performance based on likelihood ratio Chi² statistics, followed by lymphocyte and neutrophil percentages, NMR and NLR, with LMR and MLR contributing least.

Conclusions: In hospitalized patients with high cardiovascular risk, hsCRP and leukocyte-derived indices, particularly NLR and NMR, were independently associated with adverse cardiovascular outcomes. Among all parameters, hsCRP demonstrated the strongest predictive performance, underscoring its role as the most robust established inflammatory biomarker for cardiovascular risk assessment in clinical practice.

Background: Glucagon-like peptide-1 (GLP-1) is a gut-derived incretin hormone secreted in response to food-intake and pro-inflammatory stimuli. Secretion of GLP-1 leads to post-prandial insulin secretion and subsequent glucose lowering. Beyond metabolic effects, GLP-1 has pleiotropic and cardioprotective effects and GLP-1 receptor agonists reduce cardiovascular (CV) events in patients with diabetes or obesity. Direct anti-inflammatory effects are likely to contribute to beneficial effects of GLP-1RA. Clarifying the prognostic relevance of endogenous GLP-1 levels in CV disease could improve our understanding of how inflammation interacts with incretin pathways.

Methods: Circulating GLP-1 levels were measured in 888 fasted hospitalized patients in a cardiology department within a prospective single-center biobank study. The primary endpoint was a composite of non-fatal myocardial infarction, non-fatal stroke and CV death (MACE: major adverse cardiovascular events). Analyses were performed in the full cohort and stratified after individuals with and without residual inflammatory risk, defined by high-sensitivity C-reactive protein (hsCRP) levels above 2 mg/L. The association of GLP-1 with MACE was assessed using Kaplan-Meier curve, multivariable Cox proportional hazards and variable importance analyses.

Results: Median age of the cohort was 70 years, 65% were male and median GLP-1 levels were 31.5 pM. During a median follow-up period of two years, MACE was observed in 72 out of 888 patients. In Kaplan Meier curve analyses, GLP-1 levels above the median were associated with a higher rate of MACE (log-rank p=0.018). In univariable analyses, higher GLP-1 concentrations were associated with increased risk of MACE (GLP-1 above median: Hazard ratio (HR): 1.77; 95% confidence interval: 1.10, 2.85; p=0.019). These results remained significant after multivariable adjustment for age, sex, BMI, smoking, hypertension, type 2 diabetes, statin use, coronary artery disease, creatinine, LDL cholesterol and NT-proBNP (p=0.026). In variable importance analyses assessing the individual contribution of each variable from the multivariable model to prediction of MACE, GLP-1 ranked among the top predictors of outcome, outperforming traditional CV risk markers such as LDL cholesterol and creatinine. Importantly, in patients without systemic low-grade inflammation (hsCRP <2 mg/L), GLP-1 was not associated with MACE (log-rank p=0.391; HR: 1.39; 95% CI: 0.68, 2.67, p=0.393) and ranked last in variable importance analyses. Whereas in patients with hsCRP levels ≥2 mg/L, higher GLP-1 levels were strongly associated with MACE (log-rank p=0.019; HR: 2.25; 95% CI: 1.12, 4.52; p=0.023) and showed high variable importance, ranking second after NT-proBNP.

Conclusions: In hospitalized stable patients with CVD elevated circulating GLP-1 levels independently predicted MACE, with the strongest associations in those with residual inflammatory risk. These findings support the hypothesis of GLP-1 as an endogenous counter-regulatory peptide that rises in response to inflammatory activation. GLP-1 may serve as a marker of residual inflammatory risk, representing a compensatory, anti-inflammatory mechanism.