Vol 3, No 1 (2024)

The IX Spring Meeting of Young Researchers of the Italian Society of Diabetology (SID), the Italian Society of Arterial Hypertension (SIIA), the Italian Society of Internal Medicine (SIMI), the Italian Society of Cardiovascular Prevention (SIPREC) and the Italian Society for the Study of Atherosclerosis (SISA), entitled “Research drives us crazy”, was held in Rimini on February 25-27, 2024. The Congress was organized by young researchers from the aforementioned scientific societies working in the cardiometabolic field. The Congress hosted five sessions promoted by the five societies, addressing hot topics in the prevention and treatment of cardiometabolic diseases.
More than one hundred young researchers had the opportunity to discuss their scientific work in dedicated oral and poster sessions. In this conference report, we offer an overview of the key topics covered in the presentations at the meeting.

Aim. Type 2 Diabetes (DM2) is a risk factor for the development of heart failure (HF). Global longitudinal strain (GLS) is more sensitive than ejection fraction (EF) in diagnosing subclinical left ventricular systolic dysfunction (LVSD). Metabolic-associated fatty live disease has been involved in the development of subclinical LVSD-GLS. However, determinants of subclinical LVSD-GLS in DM2 remain poorly known. Our aim was to identify variables associated with altered GLS values in a cohort of DM2 individuals without heart disease and with normal EF.
Methods. The study was performed on DM2 patients (n=150) recruited in the TESEO cohort study with available data on GLS (Speckle Tracking Echocardiography, Epiq CVx Philips), hepatic steatosis (CAP), and liver stiffness (LS) (Fibroscan). Subjects with symptomatic HF, cardiovascular disease (CVD), other heart diseases, EF<50%, eGFR<30 ml/min/1.73m2, alcohol abuse, non-metabolic liver disease, and hepatic cirrhosis were excluded. Multiple regression and logistic regression analyses were used to identify GLS determinants and variables associated with subclinical LVSD-GLS (GLS≥-18%).
Results. Recruited subjects (age 61.39±7.89 years, male 57.3%) had a short DM2 duration (3.93±5.06 years) and good metabolic control (HbA1c 6.57%±1.00). Subclinical LVSD-GLS was present in 20% of subjects. Patients with LVSD-GLS had significantly higher LS values (5.77±1.75 vs 4.94±1.25, p=0.003). In multivariate regression analysis, LS values were a significant determinant of GLS, independent of age, waist circumference, diabetes duration, blood pressure, and e’ lateral. In logistic regression analysis, LS was associate with a 61% (95% CI 1.15-2,25) increased OR of LVSD-GLS independent of age, gender, WC, diabetes duration, blood pressure, ACR, LVH, and e’ lateral.
Conclusions. This study demonstrated that LS is independently associated with LVSD-GLS in DM2 patients with normal HF and without CVD. Abnormal LS values may identify a subgroup of DM2 patients at higher risk of symptomatic HF, who may benefit from closer clinical and echocardiographic monitoring.

Aims. European registries and retrospective cohorts highlighted the lack of low-density lipoprotein-cholesterol (LDL-C) goal achievement in many very high-risk patients. Hospitalized patients are often frail, and frailty is associated with all-cause mortality and cardiovascular mortality. Aim of this study is to evaluate LDL-C levels in a Real-World setting of inpatients, identify cardiovascular risk categories and highlight treatment gaps in the implementation of LDL-C control.
Methods. This retrospective, observational study included all the adult patients admitted at an Italian hospital between 2021-2022 and with LDL-C values available during hospitalization. Disease-related real-world data were collected from Hospital Information Systems using automated data extraction strategies and through the implementation of a patient-centered data repository (the Dyslipidemia Data Mart). Assessment of cardiovascular risk profiles, LDL-C target achievement according to the 2019 ESC/EAS guidelines and lipid-lowering therapies (LLT) use were performed.
Results. 13,834 patients were included: 17.15%, 13.72%, 16.82% and 49.76% were low (L), moderate (M), high (H) and very high-risk (VH) patients, respectively. The percentage of in-target patients was progressively lower moving towards worse categories (78.79% in L, 58.38% in M, 33.3% in H and 21.37% in VH). Among LLT treated patients in VH category, in-target are 28.48%; 47.6.% in H, 69.12% in M and 68.47% in L. The impact of monotherapies and combination therapies on target achievement was also analyzed.
Conclusions. This study depicts LDL-C control among an entire population of inpatients, highlighting relevant gaps especially in VH category. Future efforts must aim to reduce the cardiovascular risk of these subjects.

Aim: The interest in machine learning-based algorithms in the cardiovascular field is rapidly growing, especially for diagnostic and prognostic purposes. Recent evidence has demonstrated that certain electrocardiographic (ECG) parameters are predominantly associated with systolic function, estimated as left ventricular ejection fraction (LVEF) by echocardiography, albeit with still relatively low accuracy.
Consequently, this study aims to develop an AI-based model capable of predicting LVEF from ECG data in an Italian population.
Methods: Within the SOLOMAX project, we collected paired ECG-Echocardiography exams from 105 patients (64.82±16.02y;62.86%male). Precisely, we excluded patients with atrial fibrillation at the time of the ECG, PMK or electrostimulated rhythm, valve prostheses, previous cardiac surgery, O2 therapy or COPD, previous ablation or invasive electrophysiology procedures, currently hospitalized for Takotsubo or ACS, heart failure exacerbation, inotropic therapy, ACS over the last 3 months. We recorded anthropometric, clinical, biochemical, ECG, and Echocardiography parameters. The collected data was studied using AI-based techniques to create a new model to predict LVEF from ECG. Using an approach based on evolutionary algorithms, genetic programming was used. This approach solves a symbolic regression problem through genetic algorithms and provides a mathematical model of the relationship between ECG parameters and LVEF. The formula obtained was then used to build a simple explainable classifier, which provides a global interpretation of the link between ECG parameters and LVEF.
Results: The performance of the proposed approach and the reliability of the results were assessed using the k-fold cross-validation method and by estimating standard metrics derived from the confusion matrix associated with a binary classifier, that is, accuracy, sensitivity, specificity, precision, and F-Measure. The proposed approach consistently demonstrated its ability to distinguish patients with preserved LVEF from those with reduced LVEF. Each metric averaged across all experiments scored approximately 95%. Furthermore, in the expression generated by the AI model, the axes of the P, QRS, and T waves play a prominent role, as they are likely to provide a better interpretation of the three-dimensional cardiac geometry and, consequently, cardiac function.
Conclusions: AI applied to ECG data can be used to create cost-effective diagnostic and predictive tools for assessing LVEF. Indeed, the obtained formula highlights the relationship between ECG parameters and LVEF, as well as its complexity, which can aid in detecting heart diseases.

Aim: People with HIV (PWH) are at high risk of metabolic dysfunction-associated steatotic liver disease (MASLD), defined by the presence of hepatic steatosis plus any among overweight, diabetes, hypertension, or dyslipidemia. There are limited data whether MASLD in PWH differs in clinical presentation from MASLD in the uninfected population. Aim: to compare the severity of metabolic and hepatic dysfunction between MASLD patients with and without HIV.
Methods: 212 consecutive HIV mono-infected patients with MASLD at McGill University in Montreal were compared to a sex and age matched MASLD HIV negative control group at Policlinico Hospital in Milan. Fibroscan with controlled attenuation parameter (CAP) was used to define MASLD (CAP≥248 dB/m), severe MASLD (CAP>280 dB/m), and significant liver fibrosis (liver stiffness measurement>7.0 kPa).
Results: PWH with MASLD presented lower median BMI (28[25-31] vs 29[27-32] Kg/m2, p=0.002) and lower prevalence of obesity (26% vs 44%, p<0.001) compared to MASLD uninfected patients, along with a lower prevalence of hypertension (21% vs 38%, p<0.001). The prevalence of dyslipidemia (41% vs 26%, p<0.001), hypertriglyceridemia (26% vs 9%, p<0.001) and low HDL cholesterol (34% vs 15%, p<0.001) was higher in MASLD patients with vs without HIV. No difference in cardiovascular events and diabetes prevalence was observed between the two groups. Regarding liver disease, PWH with MASLD had lower prevalence of severe MASLD (54% vs 74% p<0.001) but higher prevalence of significant liver fibrosis (15 vs 7%, p=0.03) compared to MASLD uninfected patients. After adjustment, HIV positivity was an independent factor associated with significant liver fibrosis (figure).
Conclusions: Despite having lower BMI, PWH with MASLD have a more severe hepatic presentation and atherogenic lipid profile than MASLD uninfected patients. HIV positivity seems to be independently associated with significant liver fibrosis. Screening and follow-up for MASLD and liver fibrosis is recommended in PWH, even if they are lean.

Aim: The epigenetic modifications induced by High Fat Diets (HFDs) in long-living hematopoietic cells have been well described, but whether they affect the “aging” of neutrophils, characterized by far shorter half-life, is less clear. Neutrophils "age" through a reciprocal regulation of CXCR4, promoting a "fresh" status when leaving the BM, and CXCR2, accelerating their aging in the circulation. We study whether derailed aging exacerbates the metabolic and inflammatory consequences of HFD.
Methods: We immunophenotyped neutrophils and characterized the metabolic responses in physiology (wild-type mice, WT) and in mice with either constitutively aged neutrophils (MRP8 driven conditional deletion of CXCR4; herein CXCR4fl/flCre+) or with constitutively fresh neutrophils (MRP8 driven conditional deletion of CXCR2; CXCR2fl/flCre+), following 20 weeks of HFD feeding (45% Kcal from fat).
Results: CXCR4fl/flCre+ mice display higher plasma triglycerides levels versus WT, despite comparable glucose levels, when monitored during standard feeding. This metabolic difference was exacerbated by feeding mice a HFD for 20 weeks. Indeed, despite a comparable gluco-metabolic profile between CXCR4fl/flCre+ and WT mice, liver damage was increased in CXCR4fl/flCre+, linked to the higher accumulation of CXCR4fl/flCre+ neutrophils in the liver after 20 weeks and two hours after intragastric gavage with olive oil versus fasting. As this finding was not observed after 20 weeks of standard fat diet, these results suggest that HFD feeding redirects aged neutrophil to the liver, resulting in enriched oxidative metabolism and NETosis- and inflammation-related pathways in the liver of CXCR4fl/flCre+ mice.
Conversely, CXCR2fl/flCre+ mice were protected from obesity and insulin resistance, exhibiting a proresolutive phenotype.
In humans, increased plasma levels of Cxcl1 (ligand of CXCR2) correlated with visceral obesity and metabolic syndrome.
Conclusions: Neutrophil aging might contribute to the cardio-metabolic consequences of HFD. This aging could represent a new therapeutic target beyond the current anti-inflammatory therapies approved for the treatment of cardiovascular diseases.

Aim: To investigate BRD4-related transcriptional programmes in mouse and human models of cardiometabolic disease.
Methods: Small arteries (0.1-0.3 mm) dissected from visceral fat biopsies from healthy subjects (n=16) and patients with obesity and hypertension (n=16) were mounted on a pressurized myograph to assess the acute ex-vivo effects of BRD4 inhibition on vascular function. Vasorelaxation to acetylcholine and acetylcholine+L-NAME was evaluated, in the presence or in the absence of perivascular adipose tissue (PVAT), at baseline and after incubation with the BRD4 inhibitor RVX-208 and with selective anti-inflammatory and anti-metabolic drugs. A cardiometabolic mouse (high-fat diet+L-NAME supplementation) was orally administered RVX-208 (150 mg/kg) to test in vivo effect of chronic BRD4 inhibition. ROS and nitric oxide were assessed by confocal microscopy; protein and gene expression by Western blot and qPCR. Transcriptional changes upon BRD4 inhibition were investigated by a custom PCR array, confirmed by ChIP, and characterised by metabolomics, lipidomics and mitochondrial swelling.
Results: Endothelial-dependent vasorelaxation and vascular and perivascular TNF-alpha, IL-1beta, IL-6 were altered in cardiometabolic patients and mice. RVX-208 substantially attenuated ex-vivo vascular dysfunction, with an impact greater than anti-IL-1beta, anti-IL-6 receptor and anti-TNF-alpha. The effect was more pronounced in vessels with intact PVAT, suggesting a restoration of the PVAT anti-contractile phenotype. Gene expression profiling in PVAT unveiled hexokinase-2 (HK2) - a glycolytic enzyme implicated in mitochondrial dysfunction and inflammation - as the top downregulated gene by RVX-208 treatment. Increased binding of BRD4 to HK2 promoter in PVAT samples from cardiometabolic mice was confirmed by ChIP assays. Metabolomics assays further validated the findings by demonstrating a glycolytic shift in PVAT under disease conditions. Finally, ex vivo selective inhibition of HK2 rescued vascular dysfunction.
Conclusion: Targeting the deleterious BRD4-HK2 interplay restores cardiometabolic vascular dysfunction via reversal of the PVAT meta-inflammatory shift, highlighting a novel potential target to fight cardiometabolic pandemics.

Background: Hypertrophic cardiomyopathy (HCM) is diagnosed according to the presence of morphological and functional traits of the heart, often in the presence of genetic mutations. A specific biomarker assessing the aetiopathology of this condition is lacking. Extracellular vesicles (EVs), small particles released by all cells into biological fluids, hold promise as diagnostic and prognostic tools for cardiac diseases. We aim at characterising plasma-derived EVs isolated from 18 consecutive HCM patients and 13 healthy volunteers (CTR).
Methods: HCM underwent echocardiographic assessment and genetic testing evaluating 200 genes (NGS). EVs were isolated via ultracentrifugation from platelet-free plasma. Quantitative and qualitative assessments of EVs were performed by nanoparticle tracking analysis and transmission electron microscopy. FACS analysis was used to characterize EV subpopulations. Data are expressed as median and interquartile ranges.
Results: Most patients were male (70.8% HCM and 54% CTR) with a median age of 61 (52.5-71) years (HCM) and 47 (44-52) (CTR). Missense mutations in the MYH7 gene were the most found in HCM. The median maximum wall thickness in HCM was 16 mm (15-19) vs 8 mm (7-9) in CTR. No differences were found in EV concentrations between HCM and CTR, respectively, 3.6*109 EV/ml/cell count (2*109-5*109) and 5*10⁹ EV/ml/cell count (4*109-6*109). However, EV concentration was positively associated with the sudden cardiac death risk score in HCM (r= 0.63). Among the EVs positive for CFSE (a specific dye for EVs), those released from platelets, progenitor endothelial cells and neutrophils were increased in HCM patients vs CTR, respectively, by 1.7-, 1.5- and 1.1-fold. A strong negative association (r= -0.74) was found between progenitor endothelial cell-derived EVs and the E/E’ ratio of diastolic function, a strong predictor of first cardiac events.
Conclusions: HCM patients present a peculiar phenotypic pattern of EVs that associates which diastolic function and sudden cardiac death.

Aim: ANGPTL3 controls lipid and lipoprotein metabolism through lipoprotein lipase and endothelial lipase inhibition and the prevention of lipoprotein-derived triglycerides hydrolyzation.
Here we present the metabolic profile of ANGPTL3 deficient mice in physiology and during the development of metabolic disorders.
Methods: Angptl3 KO mice (C57BL6/J background) and their littermate controls (wild-type, WT) were fed a chow and a High Fat Diet (HFD, 60%kcal from lipids) for 16 weeks.
During the diet protocol, changes in lipids and lipoprotein profile, under fast, fed, and fast-refeed setting were assessed. The metabolic phenotype was assessed, with a Glucose Tolerance Test (GTT) and an Insulin Tolerance Test (ITT); the lipids absorption profile was assessed with an Oral Lipid Tolerance Test (OLTT).
Results: ANGPTL3 KO mice fed ad libitum a chow diet are hypolipidemic (plasma triglycerides levels: 42,42±8,80 mg/dL in ANGPTL3 KO mice compared to 122,02±55,09 mg/dL in WT mice; plasma cholesterol levels: 44,00±9,11 mg/dL in ANGPTL3 KO mice compared to 76,51±15,87 mg/dL in WT mice).
After 16h fasting, ANGPTL3 KO mice on a chow diet are hypolipidemic and display small lipoproteins less rich in cholesterol and triglycerides, as established in humans, and the same holds true for mice fed a HFD diet.
On HFD, ANGPTL3 KO mice gain less body weight, suggesting an improved metabolic profile compared to WT animals.
The hypolipidemia is conserved during all the timepoints of OLTT, both in mice on chow or HFD, suggesting a different lipid management; in spite, no significant differences in the circulating glycaemia has been proved with a GTT after 16h of fasting; likewise, a similar sensitivity to insulin has been outlined with an ITT after 4h of fasting.
Conclusions: This metabolic profiling of ANGPTL3 KO mice on chow diet or HFD highlights that these mice are hypolipidemic and may have beneficial metabolic features compared to controls.

Introduction and Aim: Several epidemiological studies indicate a strong inverse association between the risk of developing Alzheimer’s disease (AD) and plasma HDL-C levels. The mechanism by which plasma HDL influence the pathogenesis and progression of AD is still unsolved and since cholesterol esterification is a crucial step in HDL metabolism it could be involved. The purpose of this study was to evaluate cholesterol esterification and HDL subclasses in plasma and cerebrospinal fluid (CSF) of Alzheimer’s Disease (AD) patients.
Methods: The study enrolled 70 AD patients and 74 cognitively-normal controls comparable for age and sex. Lipids and lipoprotein profile, cholesterol esterification, and cholesterol efflux capacity (CEC) were evaluated in plasma and CSF using assays set for measurement in plasma, which were appropriately modified for CSF.
Results: AD patients have normal plasma lipids, but significantly reduced unesterified cholesterol and unesterified/total cholesterol ratio. Lecithin:cholesterol acyltransferase (LCAT) activity and cholesterol esterification rate (CER), two measures of the efficiency of the esterification process, were reduced by 29% and 16%, respectively, in plasma of AD patients. Plasma HDL subclass distribution in AD patients was comparable to that of controls, but the content of small discoidal preβ-HDL particles was significantly reduced. In agreement with the reduced preβ-HDL particles, cholesterol efflux capacity mediated by the transporters ABCA1 and ABCG1 was reduced in AD patients’ plasma. The CSF unesterified to total cholesterol ratio was increased in AD patients, and CSF CER and CEC from astrocytes were significantly reduced in AD patients. In the AD group, a significant positive correlation was observed between plasma unesterified cholesterol and unesterified/total cholesterol ratio with Aβ1-42 CSF content.
Conclusions: Taken together data indicate that cholesterol esterification is hampered in plasma and CSF of AD patients, and that plasma cholesterol esterification biomarkers (unesterified cholesterol and unesterified/total cholesterol ratio) are significantly associated to disease biomarkers (i.e., CSF Aβ1-42).