Vol 2, No 1 (2023)

The VIII 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) “Basic and clinical research: Until grant let us apart” was held in Rimini on April 16-18, 2023. As is customary, the Congress was organized by the young members of the aforementioned scientific societies operating in the cardiometabolic field. The Congress featured five sessions dedicated to the discussion of the recent evidence from both basic and clinical researchers in cardiometabolic treatment and prevention. Many young researchers had the opportunity to present their scientific works in dedicated oral and poster sessions. In this report, we provided a summary of the main issues discussed during the Meeting lectures.

Aim: noninvasive echocardiographic analysis of blood-tissue interaction has recently been made possible by a sophisticated mathematical model. This model uses speckle-tracking technology to estimate instantaneous intraventricular gradients (IVPGs), which are represented as hemodynamic forces (HDFs). The aim of the present study is to examine how HDFs are affected by gender and age, providing reference value.
Methods: 85 healthy subjects were recruited and underwent transthoracic echocardiography. Speckle-tracking analysis was performed from the three apical views, and the mitral annulus and left ventricular outflow tract were measured to compute HDFs. Longitudinal HDFs have been examined, decomposing them in amplitude and time parameters.
Results: study population showed a median age of 47[25-60] years and 53% were female.
Female patients showed lower LVMi (60.1±11.8mg/m2 vs. 71.4±16.8mg/m2, p=0.001), lower LVEDV (84.6±14.6ml vs. 108±20.7ml, p<0.001), and a lower E/e’ (7.26[6.47;7.78] vs. 5.31 [4.77;6.30], p<0.001). Nor systolic nor diastolic blood pressure differed between male and female patients (p NS for both). Several time parameters differed between gender: female subjects had a later systolic deceleration peak (38.7±4.21% vs. 34.8±4.31%, p<0.001) and a later diastolic deceleration peak (60.9±7.64% vs. 56.5±8.39%, p=0.015). No amplitude HDFs parameter was found to differ between gender (p NS for all).
Regarding age, patients over50 years showed higher systolic (124±15.4mmHg vs. 115±10.8mmHg, p=0.008) and diastolic (75.2±8.99mmHg vs. 69.8±7.16mmHg, p=0.005) blood pressure, and higher E/e’ (7.41[6.91;8.58] vs. 5.58[4.81;6.56], p<0.001). HDFs time variables differed between patients under and over50 years: systolic ejection duration was longer in over50-group (27.8±3.44% vs. 25.6±3.48%, p=0.005), systolic deceleration duration was shorter in over50-group (7.69±1.48% vs. 8.54±1.97%, p=0.025) and systolic acceleration peak was earlier in over50-group (13.5±2.46% vs. 14.8±2.59%, p=0.020).
Conclusion: Among healthy subjects, female patients showed later systolic deceleration peak and later diastolic deceleration peak. Subjects over50 years showed longer systolic ejection duration, shorter systolic deceleration duration and earlier systolic acceleration peak.

Aim: Statin-associated muscle symptoms (SAMS) are claimed to be frequent in clinical practice. The SAMS-clinical index (SAMS-CI) assesses the likelihood that muscle symptoms are related to statin use. We evaluated the prevalence and characteristics of muscle symptoms in hypertensive patients eligible for statin therapy according to their individual cardiovascular risk.
Methods: Observational study on 390 consecutive outpatients referred to our Centre. All patients were asked the following question: “Have you ever taken a drug/nutraceutical that you think gave you muscle symptoms?”. Patients who answered “yes” were evaluated with SAMS-CI.
Results: Mean age: 60.5±13.5 years. Male prevalence: 53.8%. Patients who have ever taken a statin (“statin+” group): 250. Patients who have never taken a statin but have taken at least one other drug (“statin-” group): 140. Prevalence of muscle symptoms did not differ between the groups (p=0.217). Age and number of drugs taken were significantly associated with muscle symptoms at multivariate analysis. A not clinically significant higher SAMS-CI score emerged in the “statin+” group (p=0.004). Localization and pattern of muscle symptoms did not differ between the groups (p=0.170). Timing of muscle symptoms onset after starting the drug (p=0.036) and timing of symptom improvement after withdrawal (p=0.002) were associated with statin therapy.
Conclusions: Prevalence of patient-reported muscle symptoms was not associated with statin therapy in our real life clinical study, confirming the growing evidence that subjective muscle-related symptoms are often misattributed to statins, while they may more likely be related to the nocebo/drucebo effect or other common undiagnosed conditions.

Aim: Increased Blood Pressure (BP) Variability (BPV) may represent an alteration in BP physiological homeostatic patterns. Most physiopathological mechanisms underlying BPV are implicated in aging. Vascular aging is associated with chronic low-grade inflammation occurring in late life, known as "inflammaging", and the hallmark “mitochondrial dysfunction” associated to stress due to age-related disorders, which in turn might contribute to higher BPV and risk of cardiovascular disease. We aimed to determine whether plasma levels of the pleiotropic stress-related mitokine Growth/Differentiation Factor 15 (GDF-15) and two inflammatory biomarkers, Interleukin 6 (IL-6) and Tumor necrosis factor receptor 1 (TNFR-1), are associated with visit-to-visit BPV in a population of community-dwelling older adults.
Methods: The study population consisted of 1,096 participants [median age 75 (72-78) years; 699 females, 63.7%] selected among community-dwelling participants aged ≥70 years from the MAPT study. Plasma blood sample was collected 12 months after enrolment and BP was assessed up to seven times over a subsequent 4-year period. Systolic BPV (SBPV) and diastolic BPV (DBPV) were determined through several indicators including the coefficient of variation (CV%) and taking into account BP change over time, the order of measurements and formulas independent of mean BP levels.
Results: Higher values of GDF-15 were significantly associated with increased SBPV (all indicators) after adjustment for demographics, body mass index, MAPT randomization group, baseline systolic BP, antihypertensive drugs, diabetes mellitus, cardiovascular and non-cardiovascular comorbidities [adjusted 1-SD increase in GDF-15: β (SE)= 0.07 (0.04), p< 0.044, for CV%]. GDF-15 levels were not associated with DBPV. No significant associations were found between IL-6 and BPV, whereas TNFR1 was only partially related to DBPV.
Conclusions: Unlike inflammation biomarkers, higher GDF-15 levels were associated with greater SBPV. Our findings support the age-related process of mitochondrial dysfunction underlying BP instability, suggesting that BPV might be a potential marker of aging.

Aim: Irisin is a hormone secreted by skeletal muscle able to improve metabolic homeostasis. Serum irisin levels are reduced in type 2 diabetes (T2D), while exogenous irisin administration improves glycemic control in diabetic mice. We have previously demonstrated that irisin promotes beta-cell survival and function both in vitro and in vivo in healthy wild type mice. We have also demonstrated that irisin restores the defective glucose-stimulated insulin secretion (GSIS) and reduces apoptosis in human pancreatic islets from patients with T2D. Nevertheless, the beta-cellular effects of in vivo irisin administration to T2D mice are still unknown.
Methods: C57Bl/6 mice (n = 8) were fed a high-fat diet (HFD, 60% of energy deriving from fat) for 10 weeks and then intraperitoneally injected with streptozotocin (STZ, 100 mg/kg) to induce diabetes. Four standard diet (SD)-fed mice were used as control. HFD/STZ mice were treated with 0.5 μg/g irisin (n = 4) or vehicle (n = 4), for 14 days. Fasting glycemia, insulinemia, body weight, glucose tolerance, and pancreatic islet function were assessed. Pancreatic islet architecture was also evaluated through immunofluorescence analyses.
Results: Compared to SD mice, HFD/STZ mice showed higher fasting glycemia and body weight, glucose intolerance, and reduced GSIS; in addition, HFD/STZ mice showed reduced islet volume (-78%), beta-cell area (-35%), and insulin content (-60%), and increased alpha-cell area (+54%). Irisin administration significantly restored glycemia (-31%), body weight (-13%), glucose tolerance (-27%), GSIS (+23%), islet volume (+61%), beta-cell area (+34%) and alpha-cell area (-49%), and insulin content (+36%). Of note, irisin induced a 9-fold increase in beta-cell proliferation rate.
Conclusions: These results show that irisin improves glycemic homeostasis and restores the functional beta-cell mass when administered in vivo to diabetic mice, probably by promoting beta-cell proliferation.

Background: Acute ischemic stroke (AIS) represents one of the principal causes of neurological morbidity and mortality worldwide. For a prompt and efficient cerebral blood restoration, intravenous treatment with rt-PA is often combined with mechanical thrombectomy (MT) which provides cerebral thrombi (CT) as study material, allowing the investigation of its cellular composition, morphological and histopathological features. Indeed, the determination of stroke etiology, typically defined by the TOAST classification, is paramount for prognostic factors, outcome, and management of the event. Aim of the study is therefore to highlight and analyze gene expression profiles in thrombotic tissue and peripheral blood (PB) in the comparison between strokes of cardioembolic (CE) and atherosclerotic (LAA) origin.
Methods: We performed gene expression profiles of 92 patients. CT were stored in RNA later and RNA was extracted by PAX gene blood miRNA kit. The global gene expression profile was assessed by Affymetrix technology using GeneChip Human Transcriptome Array 2.0 combined with Affymetrix Transcriptome Analysis Console (TAC) Software.
Results: Currently, we focused our attention on CT data analysis. The analysis revealed a significant difference (p-value<0.05 and FoldChange=2 as threshold) in gene expression when comparing LAA and CE stroke. In particular, from CT of atherosclerotic origin emerges an overexpression of 1766 genes. Prominent among them are genes such as MMP-9, TGFB, TGFBR and CXCL1, primarily involved in neutrophil-mediated immunity, Blood-Brain Barrier (BBB) disruption processes, and associated with atherosclerotic plaque instability and related to poor neurological outcome, suggesting a deleterious role in human brain injury. As concerns CE patients, 57 genes mainly involved in transcriptional regulatory processes turn out to be significantly overexpressed.
Conclusions: Transcriptome profiling is a powerful weapon for revealing expression patterns associated with complex disorders. The variation of gene expression profiles confirmed and extended several known pathophysiological mechanisms and may be one way of delineating different stroke etiology.

Introduction and Aims: Homozygous-familial hypercholesterolemia (Ho-FH) is a rare condition due to biallelic mutations in low-density lipoprotein-receptor (LDL-R) genes characterized by high level of LDL-cholesterol (LDL-c) and huge risk of premature atherosclerotic cardiovascular disease (ASCVD), determining low quality of life and life expectancy.
Lomitapide represents a therapeutic option for Ho-FH, but caution should be observed when used in fatty liver disease (FLD) and hypertransaminasemia since it is associated with onset/worsening of liver steatosis. We present a case of safe lomitapide therapy in an adult Ho-FH patient with pre-existing FLD.
Case presentation: A 39-year-old man with severe hypercholesterolemia since childhood (LDL-c 405 mg/dl) and premature coronary heart disease history, was referred to our Modena Lipid Clinic. He presented an overt metabolic syndrome, FLD with hypertransaminasemia and elastosonographic significant liver fibrosis. Lipid-lowering-therapy (LLT) included rosuvastatin 20 mg, ezetimibe and evolocumab 140 mg twice a month without reaching LDL-c goal. Genetic analysis revealed homozygous pathogenic LDL-R gene mutation. Evolocumab was increased up to 420 mg twice a month and LDL-apheresis was started with quality of life worsening. Therefore, lomitapide 5 mg daily and low-fat diet were started, obtaining weight loss and lipid profile improvement. However, liver enzymes elevation higher than 5-fold was observed, leading to lomitapide discontinuation and baseline liver enzymes values restoration. After one-month wash-out, lomitapide was gradually reintroduced up to 5 mg daily without significant hypertransaminasemia recurrence, leading to LDL-c target achievement and LDL-apheresis discontinuation. Adherence to low-fat diet and weight loss resulted in FLD and fibrosis improvement.
Conclusion: Ho-FH requires complex, combined treatment. Metabolic comorbidities co-existence makes Ho-FH management more difficult. Lomitapide can be safely used in Ho-FH patients with FLD and hypertransaminasemia, but strict follow-up of liver disease and a multidisciplinary approach are needed. Before lomitapide introduction, low-fat diet should be started advantageously and weight stabilization should be obtained.

Aim. The mainstays for the treatment of non-alcoholic fatty liver disease (NAFLD) are lifestyle intervention with the aim of significant weight loss alongside aggressive cardiovascular risk reduction. NAFLD is tightly associated with both obesity and type 2 diabetes (T2D). In people with T2D, glucose lowering agents that promote weight loss have shown a beneficial impact on NAFLD based on histological features. However, it remains unclear as to whether glucose lowering can improve NALFD in patients with T2D, independent of weight loss.
Methods. In a consecutively recruited population of 637 patients with T2D with HbA1c levels above treatment targets, DPP-IV inhibition, GLP-1RA therapy or SGLT2 inhibition was initiated, alongside lifestyle education with maintenance of exiting background glucose lowering treatment. We examined the longitudinal impact of the optimization of glycaemic control on fatty liver index (FLI) and Fibrosis score 4 (Fib-4) adjusting for changes in BMI and choice of glucose lowering regimen over a 12-month period.
Results. Change in HbA1c and change in FLI correlated significantly in a linear regression analysis after adjustment for change in BMI, age, sex, and drug class (R=0.467, p=0.031). The greatest reduction in FLI was observed in patients with the largest reduction in HbA1c (p<0.0001). The probability of improvements in FLI with optimization of glycaemic control was similar with all 3 glucose lowering agents, despite differences in weight reduction. Similar relationships were observed examining the changes in glycaemic control and Fib-4.
Conclusions. Significant reductions of HbA1c are associated with improvement in NAFLD independently from weight loss. These results suggest a prominent role for the optimization of glucose control in the management of coexistent NAFLD and T2D, especially in the ‘lean’ NAFLD and where significant weight loss may not be achieved.

Background: There is limited understanding of the pathophysiology of post-acute pulmonary sequelae in COVID-19-associated acute respiratory distress syndrome (ARDS). We aimed at investigating the association of circulating microRNAs (miRNAs) involved in post-transcriptional regulation of NLRP3-inflammasome pathways and lung radiological features among COVID-19- associated ARDS survivors.
Methods: We evaluated COVID-19-associated ARDS survivors at 4±2 months from clinical recovery. Patients were selected based on imaging pattern evolution according to chest high-resolution computerized tomography (HRCT) findings into “fully recovered” (FR), “pulmonary opacities” (PO) and “fibrosis-like lesions” (FL) according to radiological appearance. Plasma miRNA profiling was performed using real time quantitative polymerase chain reaction (RT-qPCR). The exosomal expression of NLRP3 inflammasome related miRNAs (miR-17-5p, miR-223-3p, miR-146a-5p) was evaluated.
Results: 31 patients (33% men, mean age 60±6 years, mean BMI 31.1±6.6 Kg/m2) were selected for the present study. No statistically significant differences between FR, PO and FL patterns were observed according to clinical features. NLRP3-inflammasome-related miRNAs such as miR-17-5p, miR-223-3p and miR-146a-5p were significantly up-regulated in patients with PO when compared to patients with FL. miR-146a-5p was also up-regulated in patients with FL than in FR.
Conclusions: In patients with long-term pulmonary radiological sequelae following COVID71 19- associated ARDS, a down-regulation of miRNAs inhibiting NLRP3 (miR-17-5p, miR-146a72 3p and miR-223-3p) correlated to fibrosis development in patients showing persistent hyper-reactivity to inflammatory stimulation. NLRP3-Inflammasome-related miRNAs could be a possible therapeutic target to prevent the fibrotic evolution of COVID-19-associated ARDS.

Aim: Treatment with the FXR agonist obeticholic acid (OCA) has been found to improve glucose metabolism in type 2 diabetes (T2DM) subjects with mechanisms not completely elucidated. In the gut, FXR is mainly expressed in the ileum where promotes transcription of fibroblast growth factor-19 (FGF19) having positive effects on glucose homeostasis, and maintains gut barrier integrity by regulating tight-junction (TJ) proteins expression. Herein, we evaluate whether subjects with dysglycemic conditions exhibit a down-regulation of the intestinal FXR-FGF19-TJ axis and whether treatment with OCA may revert this aberration.
Methods: Levels of FXR, FGF19 and TJ proteins and pro-inflammatory cytokines were assessed in ileal mucosa specimens collected during colonoscopy from 53 subjects subdivided according to their glucose tolerance in: NGT (n=26), prediabetes (n=12) and T2DM (n=15). Effects of OCA treatment was assessed on ileal mucosa specimens of subjects with prediabetes or T2DM cultured in absence or presence of OCA for 6h.
Results: Ileal FXR protein and mRNA levels were progressively decreased in prediabetes (-26%) and T2DM (-34%) as compared to the NGT group (both P<0.05). Ileal FXR downregulation was paralleled by lower FGF19 expression and circulating levels (both P<0.05). Additionally, we observed a progressive decrease of proteins and mRNA levels of the TJ zonulin (ZO)-1, occludin and claudin-1 (P<0.05 for all) with an activation of pro-inflammatory pathways in the ileal mucosa of subjects with prediabetes and T2DM as compared to the NGT group. OCA treatment resulted in an up-regulation of FGF19 expression and release (both P<0.01), mRNA and protein levels of the TJ ZO-1, occludin and claudin-1 and in reduced pro-inflammatory cytokines synthesis and release (P<0.05 for all).
Conclusion: FXR stimulation by OCA treatment reverts the altered FGF-19/TJ axis in subjects with prediabetes and T2DM, indicating intestinal FXR signaling as a novel target for prevention and/or treatment of T2DM.

Aim: Granulomatosis with polyangiitis (GPA) is a small vessel vasculitis. Inflammation of the vessel wall may induce multiple vascular damages. Atherosclerosis is accelerated during vasa inflammation. Metabolic profile and cardiovascular risk are far to be determined in these patients. Thus, Cardiovascular atherosclerotic disease (ASCVD) may represent a risk for patients' outcomes. The purpose is to evaluate ASCVD risk in GPA over time during disease follow-up.
Methods: We retrospectively evaluated 37 patients (22 Females, aged 51.45±17.15) who received a diagnosis of GPA (T0). Patients were evaluated at 1 (T1) and 2 (T2) year follow-up. All patients were treated with high steroid dose followed by a one-year tapering, associated to another immunosuppressant. Lipid profile included total cholesterol, HDL, LDL and Triacylglycerol. To evaluate inflammatory activity, we evaluate erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) and neutrophil to lymphocyte ratio (NLR) at the same time points. ANOVA for repeated values was used to evaluate the trend over time and Tukey's multiple comparisons test was a second step evaluation.
Results: At T1 there was an increase in total cholesterol compared to baseline (T1vsT0, p<0.05) and T2 (T1vs T2, p<0.05). Similarly, LDL (T1vsT0, p<0.05) presents the same trend, while Triacylglycerol increased in T1 compared to baseline (T1vsT0, p<0.05), but no difference there was in T2 compared to T1 or T0. No difference was found in HDL between the different time points. CRP was no different, despite a reduction being noticed. On the contrary, we found a reduction at T2 but not in T1 in ESR (T1vsT0, p<0.05) and NLR (T1vsT0, p<0.05).
Conclusion: Our data suggest that a change in lipid profile may not relate to better control of inflammation. On the contrary, the increase in the first year of follow-up should be a consequence of steroid treatment needed to spread disease control. These data may be helpful in the evaluation of both cardiovascular disease and lipid metabolism due to the connection between the two parameters with vessel inflammation. Further studies are needed to better evaluate the cardiovascular effect of vasculitis and consequent treatment.

Aim: Given the key role of the immune response during atherosclerosis and the therapeutic interest of biologics targeting human immune cells, the need of experimental models to translate molecular mechanisms and to test therapeutic approaches for atherosclerosis is continuously increasing. Here we describe the characteristics of an innovative immunodeficient mouse humanized with hCD34+ cells on an atheroprone background.
Methods: LDLR-KO mice were crossed with the immunodeficient C57BL/6J strain Rag2-KO/IL2rg-KO/CD47-KO (TKO, IMSR_JAX:025730) to generate an immunocompromised dyslipidemic mouse TKO-LDLR KO recipient of human hematopoietic stem cells (hCD34+).
Results: TKO-LDLR KO were first characterized for their immune and metabolic profile. TKO mice are deficient in mature lymphocytes and NK cells and this profile was conserved in TKO-LDLR KO mice. Under high cholesterol diet for 8 weeks, both males and females TKO-LDLR KO present monocytosis with increased levels of Ly6Chi monocytes compared to TKO-LDLR KO at standard diet, develop marked dyslipidemia (total cholesterol 870.9 and 890.1 mg/dL male and females respectively), steatosis and atherosclerosis. This profile confirms the suitability of TKO-LDLR KO mice for atherosclerosis studies. Next, we tested the impact of immune system humanization. TKO-LDLR KO pups received a low-dose irradiation (150-200 cGy) and thereafter 1,5-2 x 10^5 hCD34+ were injected with in the liver. Engraftment of human leukocytes (hCD45+) was evaluated after two months by flow cytometry analysis from tail blood. This approach allows to reconstitute between 10-30% of hCD45+, mainly B and T cells.
Conclusions: We have generated and characterized for the first time a humanized dyslipidemic TKO-LDLR KO mouse. This mouse model presents human B and T cells and could represent an important tool to investigate the impact of biologics targeted toward human targets in the context of atherosclerosis.

Aim: Dapagliflozin (DAPA), an SGLT2 inhibitor, has been shown to counteract heart failure outcomes in subjects with obesity and diabetes. We investigated the protective mechanisms of DAPA in human cardiac progenitor cells (hCPC) exposed to the conditioned medium (CM) from abdominal visceral (AV) and epicardial (E) adipose stem cells (ASC) and from AV mature adipocytes from obese subjects.
Methods: ASC and mature adipocytes were isolated from AV adipose tissue biopsies of 27 obese (Ob) and 19 non-Ob subjects (n-Ob), and from E adipose tissue biopsies of 9 Ob and 10 non-Ob subjects, respectively. hCPC were isolated from right auricle biopsies of 10 healthy non-Ob donors.
Results: Exposure of hCPC to the CM of adipose cells from Ob, but not from non-Ob subjects, induced apoptosis, c-Jun phosphorylation, and impairment of actin filaments, while these effects were not observed when hCPC were pretreated with DAPA. The CM of adipose cells from Ob compared to n-Ob subjects displayed a different pattern of cytokines. The levels of pro-inflammatory cytokines RANTES and MIP1β were increased in the CM from AV-ASC with higher BMI (p<0.05), while the levels of the cardioprotective factor GCSF in the CM of E-ASC were inversely correlated with BMI (p<0.05). SGLT2 was found to be expressed as both mRNA and protein in hCPC, and silencing of SGLT2 with a specific siRNA abrogated the capacity of DAPA to counteract the pro-apoptotic effects of the CM.
Conclusions: In human obesity, the CM of both AV- and E-ASC and mature adipocytes is characterized by pro-inflammatory cytokines that induce stress kinase activation and apoptosis in hCPC. DAPA prevents the hCPC damage induced by the CM through an SGLT2-dependent mechanism.

Background and Aims: Among patients at very high cardiovascular risk, some are more likely to experience recurrent cardiovascular events. In May 2022, an article was published in the European Heart Journal proposing different definitions of patients at extreme cardiovascular risk. However, the process of defining such patient is still ongoing and more data on its prevalence are needed. Our aims consisted in assessing the prevalence of patients at extreme cardiovascular risk in cardiological rehabilitation and in evaluating the clinical features of such patients. Furthermore, we wanted to establish how the extreme cardiovascular risk condition correlates with the functional improvement obtained during cardiac rehabilitation.
Methods: The study included 938 patients suffering from atherosclerosis who attended the cardiological rehabilitation of Niguarda Hospital in Milan. Patients classified as at extreme cardiovascular risk were compared with the remaining patients and a multivariate linear regression was performed with absolute functional improvement as the dependent variable.
Results: Among 938 patients, 26.9% belong to the category of extreme cardiovascular risk. Patients at extreme cardiovascular risk showed a higher average age (67.8 ± 10.4 vs 64.1 ± 11.1 years; p ≤ 0.001), a higher prevalence of significant comorbidities (peripheral arterial disease, cerebrovascular disease, dyslipidemia, diabetes, chronic kidney disease, hypertension) and a lower functional improvement during cardiac rehabilitation (102.9 ± 68.6 vs 138.1 ± 86.5 m; p ≤ 0.001). At multivariate analysis extreme cardiovascular risk remains a significant determinant of the absolute functional improvement at Six-Minute Walking Test obtained during cardiac rehabilitation with b = -0.137 and p = 0.035, together with female sex (b = -0.136; p = 0.035).
Conclusions: Extreme cardiovascular risk is a widespread condition among patients with chronic coronary syndrome and adversely affects the patient’s functional improvement during cardiac rehabilitation. The identification of patients at extreme cardiovascular risk is a goal to be pursued in order to intensify secondary prevention strategies.

Background: Obesity-related fat accumulation is linked to the metabolic syndrome and increases the risk of CVD by involving FFA, insulin resistance, and inflammation. Taking into account the findings from the third chapter, our goal was to assess the potential role of ASGR1 in metabolic reprogramming and immunoinflammatory state during obesity.
Methods: After 20 weeks of high fat diet, flow cytometry, proteomics, lipid profile, glucose tolerance, and insulin tolerance were assessed in WT and ASGR1-/- mice (HFD). Additionally, metabolic parameters such as oxygen consumption, CO2 production, and food intake were measured during the diet.
Results: After 20 weeks of HFD, the ASGR1−/− mice displayed a significant reduction in the circulating monocytes compared to WT. The body weight and food intake were comparable in between two groups. The adipose tissue VAT was significantly increased in ASGR1-/- compared to WT mice (WT 3.2%±0.8%, ASGR1-/- 4.7%±1.2%, P-value<0.001). The proteomics revealed, n=3412 proteins were aligned from which 624 proteins were significantly differentially expressed on the liver of ASGR1-/- and WT mice under HFD. From prediction analysis the significant proteins that were increase in the liver of ASGR1-/- mice were necrosis, apoptosis, and inflammation compared to the WT. Additionally, a significant downregulation in proteins protein expression involved in fatty acid synthesis and fatty acid uptake, except the increased expression of fatty acid coenzyme A ligase (FATP5), which belongs to very long chain acyl-CoA synthetases, capable mediation the transport of long chain fatty acids.
Conclusion: Our findings indicate that ASGR1 deficiency causes increased inflammation and changes in metabolic pathways when subjected to HFD. This can also have an impact on the synthesis of apolipoproteins secreted in plasma.

Background and Aim: Familial Chylomicronemia Syndrome (FCS) is a rare monogenic autosomal recessive disorder of lipid metabolism determining severe hypertriglyceridemia (HTG). As the use of Volanesorsen, a novel FCS treating drug, has been associated with thrombocytopenia, the relationship between FCS and low platelets counts should be firmly established. It has been reported also kidney complication in FCS, but the data are sparse. To this aim, we have retrospectively evaluated the spontaneous variation of platelet counts and Kidney impairment in a cohort of patients with FCS.
Methods: Single-center retrospective cohort study on 20 FCS patients included in the LIPIGEN. Medical charts have been revised to collect retrospectively information on kidney function in a cohort of patients with FCS.
Results: Across the study population, the median PLT count was 187,225 platelet/mcL. The median on treatment TG levels in the whole cohort was 1309 mg/dl. During follow-up, 8 (44.4%) patients experienced at least one episode of mild and 1 (5%) of moderate thrombocytopenia. None had severe thrombocytopenia. Mean triglycerides do not significantly predict mean platelet values. However, when considering a multivariate model including mean triglycerides, sex, the presence of hepatic steatosis and age we found that male sex and the presence of ultrasound estimated hepatic steatosis were associated with significantly lower platelet (respectively β-0,473, P=0,044 and β-0,469, P=0,048). Age was of borderline statistical significance (β-0,388, P=0,087). Across the study population, the median GFR values was 99.5 ml/min. Median eGFR was significantly associated with history of hypertension (β-0,508, P=0,031). Overall, proteinuria occurred in 5 (25%) patients, and it did not associate with hypertension, diabetes, age, sex nor triglyceride levels. Four (20.0%) patients meet the criteria of hyperfiltration whereas 3 (15.0%) were exhibiting an eGFR below 90 ml/min. Among hyperfiltrating, two had also proteinuria in at least one occasion during life. One patient with eGFR below 90 ml/min and proteinuria had a biopsy-proven diagnosis of glomerulonephritis. Overall, the impairment in kidney function was independent from age, diabetes, hypertension, median TGs, AP, sex.
Conclusions: The present analysis confirmed that thrombocytopenia and kidney impairment might be a clinical characteristics of FCS phenotype. Further studies in larger cohort are needed to better clarify if kidney disease and thrombocytopenia might be a hallmark of FCS in broader population and understand the potential patho-physiological mechanism.

Aim: Impairment of cholesterol homeostasis is one of the multiple etiopathological mechanisms at the origin of both cardiovascular and neurodegenerative diseases. The PCSK9 protein, known for its role in the degradation of hepatic LDLR and plasma cholesterol regulation, is expressed also in the CNS, where it exacerbates -amyloid neurotoxicity and reduces neuronal cholesterol uptake, suggesting an involvement in AD. This study proposes an in vitro screening of molecules (MR) with inhibitory activity on PCSK9, selecting the best compounds to test their activity on cerebral cell models and their in vivo tolerability.
Methods: 30 newly synthesized compounds were tested at increasing concentrations on human hepatoma cells (HepG2) to evaluate their cytotoxicity and efficacy in inhibiting PCSK9. MR-3 was tested on human neuroblastoma cells (SH-SY5Y) overexpressing PCSK9 to assess neurotoxicity and cholesterol uptake. Cytotoxicity was determined through MTT assay; PCSK9 secretion was quantified with an ELISA kit; and radioisotopic techniques measured cholesterol uptake . Three compounds were selected to be tested in vivo on C57BL/6 mice at a dose of 40 mg/Kg for 7 days to evaluate: tolerability with SHIRPA test; plasma lipid profile by ELISA assay; biodistribution in plasma and brain through LC-MS/MS.
Results: Among the tested compounds, MR-3, MR-532, MR-533 demonstrated no sign of cytotoxicity and the greatest efficacy on HepG2 cells (IC50=1.7μM; 5.7μM; 6.1μM). Neuronal cholesterol uptake was restored after treatment with MR-3 at 10μM (p<0,05). MR-3, MR-532, and MR-533 exhibited good in vivo tolerability; MR-3 and MR-532 were detected in plasma and brain tissue.
Conclusions: Preliminary in vitro screening allowed the identification of MR-3, MR-532, MR-533 as promising PCSK9 inhibitors. The outcome of MR-3 on neuronal cholesterol uptake may suggest a neuroprotective effect to be further investigated. In vivo treatment with selected inhibitors shown absence of toxicity, however, it is necessary to bring proof of efficacy.

Aim: Epidemiological studies, Mendelian randomized studies, and genome-wide association studies confirmed that elevated lipoprotein(a) [Lp(a)] concentration is an independent risk factor for cardiovascular diseases. However, no approved therapy for patients with elevated Lp(a) levels is available. Our aim is to investigate to what extent PCSK9 inhibitors (PCSK9i), statins, and ezetimibe affect Lp(a) level.
Methods: This meta-analysis was conducted according to the PRISMA guidelines. Databases were searched from inception to February 2023. Inclusion criteria were: (1) randomized controlled trials (RCTs) in adults (≥18 years), phase II, III or IV; (2) English language; (3) reporting the effects on Lp(a) levels; (4) with intervention duration more than 3 weeks. Pooled estimates were assessed by a random-effects model. Between-study heterogeneity was tested and measured by Cochrane’s Q test and I2 statistics.
Results: Overall, 51 RCTs were included for PCSK9i (39,271 participants), 35 RCTs for statins (15,425 participants), and 14 RCTs for ezetimibe (5,607 participants). Starting from a baseline Lp(a) level of 33.12 mg/dL, participants treated with PCSK9i compared to placebo experienced an additional reduction in Lp(a) levels of -26.34% (95%CI -28.83 to -23.85). Lp(a) levels were marginally reduced by statins by -3.43% (95%CI -9.09 to 2.23) from a baseline Lp(a) level of 15.87 mg/dL, although this reduction was not statistically significant. Finally, ezetimibe had a negligible and still not statistically significant effect on Lp(a) levels (0.51% [95%CI -1.67 to 2.70]), from a baseline Lp(a) level of 20.80 mg/dL.
Conclusions: Among the lipid-lowering approaches evaluated, only PCSK9i seemed to lower Lp(a) levels. Further research is requested to understand whether it translates into a clinically relevant cardiovascular benefit.