Vol 4, No 3 (2025)

Background: Low-Density lipoprotein (LDL) cholesterol is one of the most relevant CardioVascular (CV) risk factors. Very low therapeutic targets have been set by guidelines in the secondary prevention setting in order to reduce the risk of ischemic event recurrence. However, many studies demonstrate that these targets are largely unreached in the real-life setting, particularly in the higher cardiovascular risk classes. Our aim was to evaluate LDL target achievement in very high and extreme CV risk patients during a Cardiac Rehabilitation (CR) program.
Methods: A total of 940 patients with recent acute coronary syndrome or a diagnosis of chronic coronary syndrome who participated in a CR program were enrolled between January 2012 and December 2023 at the Niguarda Hospital (Milan). For each patient, LDL and Lipid Lowering Therapies (LLT) were evaluated at the beginning and at the end of the CR program, together with anthropometric, clinical, biochemical, and instrumental parameters. LDL targets were considered <70 mg/dL for patients before August 2019, <55 mg/dL after 2019 and <40 mg/dL for extreme CV risk subjects.
Results: Mean age was 66.9±0.6 years, 82.9% of the subjects were males, and LDL cholesterol changes from 107.3±39.3 to 64.5±24.6 from the beginning to the end of CR. At CR discharge, 88% of the subjects were on high-intensity statin (atorvastatin or rosuvastatin) therapies, and 38.1% were on ezetimibe, while only 4.6% of the subjects were treated with PCSK9 inhibitors and 0.9% with bempedoic acid. 53.1% of the patients reached the LDL therapeutic target with particularly positive peaks in 2018 (72.8%, the year before the release of the latest dyslipidaemia guidelines that reduced the target) and 2022 and 2023 (78.8% and 75.7% respectively). 29.8% of the patients had extreme CV risk, they achieved the target of LDL <40 mg/dL only in 16.4%, with a higher prevalence in the latest years (32% in 2022 and 22.7% in 2023).
Conclusions: Our results demonstrate a higher achievement of LDL cholesterol target in a secondary prevention program when compared to previous observational studies. The longer distance from guidelines publication, together with the new pharmacological treatment, could be the reason for these positive results. However, more attention should be paid to extreme CV risk both in terms of identification and treatment.

Background and aims: The miRNomic and lipidomic profiles of mice with specific genotypes/phenotypes were integrated with a novel approach, with the aim of increasing our knowledge on the mutual interplay between miRNAs and lipids, and thus to discover previously uncharacterized miRNAs, potentially playing a role in lipid metabolism.
Methods: miRNomic and lipidomic analyses were performed in wild-type, Pcsk9 and Ldlr knockout mice fed normal laboratory diet or Western diet. Small RNA was extracted from liver, brain, duodenum, jejunum, ileum and abdominal white adipose tissue and quantified by RNAseq. Lipid species were quantified by high-throughput mass-spectrometry in liver, aorta and plasma. miRNA expression levels were tested for correlations with each lipid measurement in different samples. Highly correlated, uncharacterized miRNAs were subjected to testing in vitro in murine hepatoma Hepa1-6 cells. For each miRNA to be tested, cells were transfected with the miRNA mimic, the miRNA inhibitor and a non-target control. After 24-hours incubation, the cellular content of cholesterol and triglycerides was measured. For each miRNA, at least three independent experiments were carried out.
Results: Correlation analyses between miRNA expression levels and lipid concentrations in the different experimental conditions led to the selection of miRNAs potentially playing a major role in the regulation of lipid levels. Correlations mainly clustered in liver. Among selected miRNAs, some were already known to be related to lipid metabolism (miR-33, miR-210 and miR-21a) whereas others, including miR-431-5p, miR-434-3p, miR-434-5p and miR-677-5p had never been associated to lipid changes before. In vitro experiments allowed to highlight a potential role of miR-431-5p and miR-677-5p in the modulation of total cholesterol and triglyceride concentrations.
Conclusions: This study, bridging miRNomic and lipidomic data in well characterized mouse models, allowed to identify novel miRNAs potentially playing a role in the modulation of lipid levels.

Background and aims: Acute lymphoblastic leukaemia (ALL) is the most common haematological cancer in children. In high-risk cases, treatment involves chemotherapy, radiation and stem cell transplantation. Despite high survival rates (up to 90%), increased cardiovascular and metabolic risk has been observed in survivors. This project aims to study post-transplant metabolic and inflammatory changes and their impact on mature immune cells derived from stem cells.
Methods: 14 ALL survivors who underwent transplantation (11 males, 3 females) and 14 matched sibling donors (3 males, 11 females) were selected according to defined criteria. Each recipient-donor couple were profiled for medical anamnesis and clinical assessment and were subjected to a blood collection, for immunophenotyping, transcriptomic analysis and telomere length measurement. An ultrasound examination of the supra-aortic trunks was also performed to assess intima-media thickness (IMT).
Results: No differences have been detected in anthropometric characteristics, however, signs of dyslipidaemia were observed in recipients (cholesterol: 157.5 SE9.194 vs 171.9 SE10.43; triglycerides 65.57 SE5.216 vs 105.0 SE11.31; hyperinsulinemia 8.457 SE1.870 vs 15.23 SE2.945 and an increase in the HOMA index (1.587 SE0.3653 vs 2.981 SE0.6225). Metabolic syndrome was diagnosed in 14.3% of recipients. Carotid IMT analysis showed accelerated thickening (0.0083 vs 0.0034). Plasma proteomic analysis revealed increased levels of pro-inflammatory proteins (CRP, SAAP) and proteins related to dyslipidaemia (APOB, APOC4-APOC2).
This metabolic and inflammatory phenotype is associated with accelerated telomere shortening in circulating immune cells (slope -0.009 vs -0.0008), a reduction in CD34+ and an increase in circulating CD19+. RNA-seq confirmed inflammation and B-cell dysfunction. Plasma IgM immunoglobulins were decreased in recipients compared to donors (58.62 SE4.062 vs 49.23 SE3.188), while IgG levels were comparable (350.6 SE14.49 vs 329.7 SE16.43).
Conclusions: These data suggest that TBI conditioning negatively impact on the immunometabolic profile of cALL survivors, contributing to an increased risk of long-term cardiovascular complications.
Together this evidence suggests the need to optimize the clinical follow-up strategies to mitigate the increased cardiometabolic risk in cALL survivors.

Background and aims: Apolipoprotein E (ApoE) is a key regulator of lipid metabolism, controlling transport and clearance of triglyceride rich lipoproteins. Although ApoE is produced in several tissues, the liver, especially hepatocytes, is the main source of circulating ApoE. Whole body ApoE knockout mice develop severe dyslipidemia, whereas mice lacking ApoE only in hepatocytes (ApoEΔHep) display only mild alterations on a standard diet, suggesting compensation from extrahepatic sources and raising questions about the specific contribution of hepatic ApoE. This work investigated the distinct role of hepatocyte derived ApoE in hepatic lipid handling.
Methods: ApoEΔHep mice were generated by crossing ApoE flox/flox mice with Albumin Cre mice. Animals were kept on a standard chow diet for 12 weeks. Lipid metabolism was assessed by FPLC, Western blotting, hepatic gene expression analysis and functional assays of lipoprotein production and clearance.
Results: Plasma WB confirmed that hepatocyte-derived ApoE is the main circulating source of the protein, which was absent in ApoEΔHep mice. Total plasma cholesterol was similar to controls under both standard diet (76,199,99 vs. 85,6415,20 mg/dL) and high cholesterol diet (154,726,45 vs. 151,414,35 mg/dL). However, lipoprotein profiling revealed a shift toward LDL enriched cholesterol in ApoEΔHep mice, in contrast to the VLDL dominant pattern of global ApoE KO mice. ApoEΔHep animals showed delayed clearance of triglycerides, consistent with impaired removal of triglyceride rich lipoproteins, and a 46% reduction (p < 0.0001) in VLDL production, indicating a role for hepatocyte ApoE in the synthesis and secretion of these particles. Hepatic transcript analysis pointed to deregulated lipid metabolic pathways.
Conclusions: This work highlights a dual role of hepatocyte-derived ApoE in both the production and clearance of TG-rich lipoproteins, pointing to distinct function of ApoE at systemic versus hepatocellular level. Ongoing molecular analyses aim to further dissect the intracellular mechanisms of lipoprotein synthesis and clarify the hepatocyte-specific contributions of ApoE to lipid metabolism and dyslipidemia.

Background and aim. Since increased plasma ceramide levels have recently emerged as a pro-atherogenic factor, selective modulation of hepatic ceramide biosynthesis might represent a novel therapeutic approach to reduce cardiovascular risk.
Methods. The efficacy of siRNA-based treatments (siPOOLs) on ceramide level modulation was tested by targeting single enzymes or couples of enzymes playing key roles in ceramide metabolism. In vitro, the effects of siRNAs on target gene expression and lipidome changes were evaluated in Hepa1c1c7 murine hepatocytes (96 hours after treatment, 6 nM). For in vivo studies, siPOOLs were encapsulated in lipid nanoparticles (LNPs) and first tested for hepatic tropism by confocal microscopy (single intraperitoneal injection of Cy5-labelled LNPs, 2 mg/kg, in C57Bl/6 male mice). In vivo gene silencing efficacy, effects on plasma and liver lipidome, and histological analyses were assessed after four intraperitoneal injections (2 mg/kg every 72 hours) in 8-week-old C57Bl/6 male mice.
Results. In vitro validation identified siRNAs effective in reducing target gene expression and lowering ceramide levels in hepatocytes. Biodistribution studies showed that siRNA-LNPs accumulated primarily in the liver and in the spleen. In vivo testing showed variable silencing efficacy of siRNA-LNPs, ranging from –17% (Sptlc1) to –80% (Sptssa), depending on the target and the combination/dose administered. Despite a promising silencing effect, lipidomic analyses of plasma and liver did not show effective modulation of ceramide levels. Treated mice displayed extramedullary hematopoiesis in the spleen and, in a few cases, hepatic necrosis.
Conclusions. The siRNA-LNP approach resulted in efficient silencing but did not translate into plasma ceramide modulation. A second strategy, consisting of N-acetyl galactosamine-conjugated siRNAs, will be tested in vitro and in vivo, with the aim of achieving higher efficacy and reduced toxicity.

Background: Atherosclerosis represents one of the main cardiovascular risk factors and is driven by high plasma cholesterol levels and an impaired immuno-inflammatory response. Despite effective lipid-lowering therapies, some patients exhibit persistent residual inflammatory risk, highlighting the need for experimental models that enable the translation of molecular mechanisms and immunomodulatory cardiovascular therapies from preclinical to clinical studies.
In this context, we present the immuno-metabolic characterization of an immunodeficient mouse model with an atheroprone genetic background, whose immune system is reconstituted with human hematopoietic stem cells (CD34+).
Metodi: Humanized TKO-LDLr KO mice (obtained by crossing LDLr KO mice with immunodeficient Rag2-KO/IL2rg-KO/CD47-KO mice, HuTKOL) were generated by irradiating 2-3-day-old pups at low doses (250cGy), followed by intrahepatic injection of commercial human CD34+ cells or those derived from iPSCs (250,000-300,000 cells/mouse). After 12 weeks, human cell engraftment was assessed by FACS. The HuTKOL mice were then fed a high-cholesterol Western-type diet (WTD) for 12weeks to study the immuno-metabolic phenotype and atherosclerosis development.
Risultati: HuTKO-L mice showed good engraftment of commercial human CD34+ cells in circulation (%hCD45+/total live leukocytes: 40.97%,SE±3.46%). B-lymphocytes were the most abundant population at 8 weeks (%hCD19+/hCD45+: 72.81%,SE±2.33%), but decreased progressively (%hCD19+/hCD45+: 13.85%,SE±2.98%), while T-lymphocytes showed the opposite trend, becoming the dominant population after 12 weeks of WTD (%hCD3+/hCD45+: 58.84%,SE±4.86%).
Exposure to WTD induced dyslipidemia (plasma cholesterol: 1086.90mg/dl,SE±65.15) and atherosclerosis (%plaque obstruction in the aortic root: 24.79%,SE±3.17%; atherosclerotic lesion volume: 0.32mm³; %fibrosis/plaque area: 33.94%,SE±16.17%), with infiltration of human immune cells into the plaques. Furthermore, WTD feeding induced the expansion of CD4-memory T-lymphocytes and the production of IgM against atherosclerosis-associated antigens.
Characterization of iPSC-derived CD34+ cells and their engraftment in TKO-L mice is currently ongoing.
Conclusioni: HuTKOL mice represent a tool for studying the dynamics of human adaptive immunity under dyslipidemic conditions and for testing immunomodulation strategies in cardiovascular diseases.