Vol 4, No 2 (2025)

Introduction and aim. Obesity and metabolic dysfunction-associated steatotic liver disease (MASLD) are global health concerns linked to an increased risk of atherosclerotic cardiovascular disease (ASCVD). Despite progresses in elucidating relevant pathophysiological mechanisms involved in obesity and MASLD, a comprehensive understanding toward ASCVD risk is missing due to the lack of reliable in vitro models reproducing obesity-related dysfunctional adipose tissue and MASLD.
This study focuses on developing and characterizing innovative in vitro models recapitulating key features of these conditions assessing novel molecular signatures for potential drug targeting.
Methods. Human-derived SW872 adipocytes, dysfunctional SW872 (SW872-OA) obtained by 7- days 100 μM oleic acid (OA), 17-days spontaneously differentiated SW872 (SW872-AUTO), human hepatoma HepG2 cells, and a HepG2-based steatotic-like model induced with 7-days 100 μM OA (HepG2-OA), were selected. All in vitro models were characterized using ¹H NMR spectroscopy, light-microscopy, spectrophotometry, flow cytometry, and RT-qPCR.
Results and Discussion. Metabolomic and lipidomic analysis allowed to identify specific metabolite signatures associated with dysfunctional patterns in both SW872 and HepG2 cell models. SW872- OA, SW872-AUTO, and HepG2-OA models showed increased lipid/triglycerides accumulation, confirmed by light-microscopy observations, and spectrophotometry quantification. HepG2-OA cells exhibited elevated triglyceride synthesis genes expression. Functional assays revealed reduced glucose uptake and elevated ROS production across all dysfunctional models. Trolox antioxidant treatment mitigated ROS levels in SW872 and HepG2 cells with minor effects on SW872-OA, SW872-AUTO, and HepG2-OA. Gene expression analysis (GEA) showed significant upregulation of oxidative stress-related genes in HepG2-OA cells. GEA in SW872-OA and SW872-AUTO cells revealed upregulation of genes involved in adipocyte differentiation, inflammation, and glycemic homeostasis.
Conclusion. These results showed that OA-treatment and SW872-spontaneous differentiation generate in vitro models recapitulating dysfunctional adipose tissue and MASLD.
Relevance for AtheroNET. Dysfunctional SW872 and HepG2 cell models show specific molecular, metabolomic and lipidomic signatures which may translate to innovative pharmacological targets for obesity, MASLD, and ASCVD.

Introduction and Aim: Inflammation plays a central role in the development and progression of atherosclerosis. Pro- resolving lipid mediators, including Resolvins (RvE1) and Maresins (MaR1) are key regulators of the resolution phase of inflammation. Despite their known anti-inflammatory effects, their impact on nitric oxide (NO) and prostacyclin (PGI2) levels in human umbilical vein endothelial cells (HUVECs) remains underexplored. This study investigates the effects of RvE1 and MaR1 on vascular inflammation, focusing on NO and PGI2 in HUVECs.
Methods: An in vitro inflammatory model was established by incubating HUVECs with lipopolysaccharides (LPS, 100 µg/mL) and interleukin-1 beta (IL-1β, 100 ng/mL) for 24 hours. RvE1 and MaR1 (100 nM) were then applied under the same conditions. Levels of tumor necrosis factor-alpha (TNF-α), total nitrite-nitrate, and the stable PGI2 metabolite 6- keto-PGF1α were measured in the incubation medium using ELISA.
Results and Discussion: LPS and IL-1β significantly increased TNF-α levels, confirming the inflammatory response. RvE1 and MaR1 significantly reduced nitrite-nitrate levels, suggesting their role in mitigating vascular inflammation. However, no significant changes were observed in 6-keto-PGF1α levels (n=3-4, p>0.05). These findings highlight the selective effects of RvE1 and MaR1 on different pathways involved in endothelial inflammation.
Conclusion: This preliminary study demonstrates that RvE1 and MaR1 appear to reduce nitrite-nitrate levels, their lack of impact on 6-keto-PGF1α suggests distinct pathway-specific actions that warrant further investigation. Future studies with larger sample sizes, varied inflammatory conditions, are essential to confirm these findings and better understand their therapeutic potential.
Relevance to AtheroNET: This research aligns with AtheroNET's goal of exploring novel anti-inflammatory strategies, emphasizing the importance of pro-resolving lipid mediators in vascular health.

This work was supported by funding from Scientific and Technological Research Council of Türkiye (TÜBİTAK) (Project number: 1919B012313469)

Introduction and aim: Cardiovascular disease (CVD) and type 2 diabetes (T2D) are interconnected chronic conditions which cause significant global health challenges and mortality. T2D is a risk factor for CVD characterized by insulin resistance and beta-cell dysfunction. This study aims to investigate the association between the blood plasma proteome and T2D in the Trøndelag Health (HUNT) Study.
Methods: The HUNT Study is a population-based cohort with four waves of enrollment beginning in 1984. 5,402 samples from HUNT3 were analysed with SomaScan including 3,221 on SomaScanv4.0 (~5,000 proteins) and 2,181 on SomaScanv4.1 (~7,000 proteins). T2D was defined based on self-reported disease from questionnaires. Binary logistic regression analysis was performed to test the associations of protein concentrations with T2D. Models included adjustment by sex, age, waist-hip-ratio (WHR), smoking status, and comorbidities.
Results and Discussion: We identified 584 proteins that are significantly associated with T2D. From these proteins, CILP2 and MXRA8 are associated with decreased risk, while PLXB2 and NFASC are associated with increased risk for having T2D. The most significant pathway from GO enrichment analysis includes proteins related to lipid catabolic process (FDR adjusted p- value: 0.000054). Indeed, several proteins significantly associated with T2D are known for their role in lipid metabolism, for instance Adiponectin (OR 0.55; 95% CI 0.46 - 0.65), Apo A – IV (1.57;1.37 - 1.81), Apo B (0.63; 0.55 - 0.73), Apo C – I (0.73; 0.64 - 0.84), and Apo D (0.49;0.38 - 0.63).
Conclusion: The proteins and pathways identified represent insights into the underlying molecular mechanism of T2D and may serve as potential biomarkers for risk prediction and prevention for CVD.

Relevance for AtheroNET: The findings suggest that early identification of these proteins could mitigate cardiovascular risks in individuals with or at risk for T2D.

Introduction and aim. The interplay between nutrition patterns, gut microbiota-derived metabolites and cardiovascular diseases (CVDs) has recently become a field of intensive research. Gut microbiota-derived metabolites represent an attractive source of biomarkers for CVDs, including atherosclerotic cardiovascular disease (ASCVD) and metabolic diseases. Trimethylamine (TMA), a gut-derived metabolite, undergoes oxidation in the liver, resulting in trimethylamine N-oxide (TMAO). Recent evidence from in vivo studies and clinical trials has correlated TMAO with increased CVD risk and occurrence due to the atherogenic potential of TMAO. TMAO has been quantified by exploiting mass spectrometry (MS) techniques, coupled either with liquid (LC) or gas chromatography (GC), and nuclear magnetic resonance (NMR) spectroscopy. However, standardization parameters and conditions still represent a critical issue when dealing with data reproducibility and robustness.
Hence, this study aimed at developing a validated and highly reproducible method for the quantification of TMAO in human-derived plasma.
Methods. A high-performance LC (HPLC) system (Shimadzu) coupled to tandem MS (MS/MS) (Sciex QTRAP 6500+) was selected, along with multiple reaction monitoring (MRM) modality and electrospray ionization (ESI) in positive polarity. Calibration curve was obtained spiking a healthy subject serum sample with different TMAO concentrations in the ng/mL range.

Results and Discussion. The HPLC-MS/MS method was optimized according to the different MS parameters and by selecting the most appropriate column for HPLC assessment. Method validation was performed evaluating the intra-/inter-assay accuracy and precision. The coefficient of variation (CV%) always resulted below 20%.
Conclusion. The HPLC-MS/MS method showed to be robust and reproducible according to standardization requirements, and useful to assess ASCVD risk in a cohort with subclinical atherosclerosis, food intake record and microbiome evaluation¹.
Relevance for AtheroNET. TMAO data from characterized CVD patient cohorts, obtained in a reproducible and standardized manner, could be integrated by artificial intelligence/machine learning-based approach to improve CVD risk stratification toward precision medicine.