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An aortic aneurysm is a ruinous condition that compromises the aortic wall. If inaccurately monitored, it could evolve in an extensive dilatation of the vessel and eventually in the aorta's devastating rupture with low survival rates. The recent attempts to increase clinical remission and disease clearance have only partially alleviated the disease-related poor prognosis. Indeed, aortic aneurysm events annually increase their impact on the National Health Systems of industrialized countries. In recent years, liquid biopsy tests have gained attention as a valuable alternative to traditional diagnostics. Classified as small extracellular vesicles of endosomal origin, exosomes are 30-150 nm diameter vesicles, which are physiologically produced by all cell types and secreted through an exocytosis process in blood, urine, cerebrospinal fluid, and other body fluids. Representing the molecular footprint of the cells of origin, exosomes have been recently considered to design a reliable liquid biopsy for non-invasive monitoring of disease evolution and a valuable tool to monitor the therapy response. Metalloproteinases have been reported as specific markers of aortic aneurysm in terms of disease staging and progression. Of interest, it has been demonstrated a specular metalloproteinase expression in the extracellular vesicles of patients, thus constituting a personal "barcode" of disease progression. This evidence could potentially allow the definition of innovative liquid biopsy approaches for monitoring the disease together with a comprehensive aortic aneurysm-derived exosomes molecular characterization and trapping.

Introduction: Dyslipidemias are common clinical conditions associated to cardiovascular diseases. Among these, Familial Hypercholesterolemia (FH) and severe Hypertriglyceridemia (sHTG) are the most frequent ones. The aim of this study is to evaluate the possible use of a wide next-generation-sequencing (NGS) panel of 28 genes involved in lipid metabolism, to improve the molecular diagnosis of dyslipidemias. Methods: A reanalysis of 25 patients (21 FH and 4 sHTG) previously analyzed for a few causative genes has been carried on. Patients bearing different types of variants [single nucleotide variants (SNVs) and copy number variations (CNVs)] in different genes, previously analyzed with Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA) have been selected. DNA libraries have been prepared using Agilent SureSelect target enrichment protocol; the sequencing has been performed using Illumina MiSeq (V2 150x2 Micro). The results of the sequencing have been evaluated by Agilent SureCall and Agilent Alissa Align & Call and Intrepret pipelines. Results: All previously identified SNVs and CNVs have been confirmed by NGS. Additional rare variants, not always associated with dyslipidemias, were found in 23/25 patients. An additional pathogenic variant in the APOB gene has been identified in a sHTG patient carrying only 1 pathogenic variant in the APOA5 gene (causative of sHTG). Conclusions: The NGS-method confirmed all the results obtained with direct sequencing and MLPA methodologies. Additional rare variants were detected, even if most of them turned out to be variant of uncertain significance (VUS). In conclusion, this NGS approach may enhance the molecular diagnosis of different types of dyslipidemias, thereby leading to a better understanding and detection of complex phenotypes.

Introduction: atherosclerosis is one of the leading causes of death and morbidity worldwide. It consists in the development of plaques in the intima media layers of arteries due to lipid accumulation and oxidation, causing massive inflammation. We aim to better understand the role of Galectin-3 (Gal-3) and Lipoprotein(a) [Lp(a)] as possible peripheral markers of plaque presence. Methods: Gal-3 and Lp(a) were measured in plasma samples from 99 patients undergoing carotid endarterectomy and 78 healthy controls, by immunometric assays. Plaques were classified histologically, according to the American Heart Association (AHA) guidelines as type Va (fibroatheroma), Vb (mainly calcific) and Vl (complicated lesion). Results: Gal-3 and Lp(a) plasma levels are higher in patients compared to controls [19.8 ng/mL (SD 5.8) vs 14.0 ng/mL (3.6)], p

OBJECTIVE: Autosomal dominant hypercholesterolemias are due to defects in the LDL receptor (LDLR) gene, in the apolipoprotein B-100 gene or in the proprotein convertase subtilisin/kexin type 9 gene. The aim of this study was to identify and functionally characterize mutations in the LDLR gene that account for most cases of familial hypercholesterolemia (FH). METHODS: We enrolled 56 unrelated patients from Southern Italy with a clinical diagnosis of FH. The mutation screening was performed by direct sequencing of the promoter and the 18 exons of the LDLR gene and by multiplex ligation-dependent probe amplification (MLPA) analysis to search for large rearrangements. RESULTS AND CONCLUSION: We found 5 new mutations, the causative role of which was demonstrated by functional characterization performed by quantification of fluorescent LDL uptake in EBV-transformed B lymphocytes. These results enlarge the spectrum of FH-causative LDLR mutations. Lastly, screening for large rearrangements is highly recommended for the genetic diagnosis of FH.

Background and Aims Familial hypercholesterolemia (FH) is an autosomal dominant disease that leads to cardiovascular (CV) disease. Proprotein convertase subtilisin/kexin type 9 inhibitors (PCSK9-I) demonstrated efficacy in low-density lipoprotein cholesterol (LDL-C) reduction and in prevention of CV events. The aim of our study is to evaluate the relationship between LDL receptor (LDLR) mutations and response to PCSK9-I therapy. Methods and Results We evaluated total cholesterol (TC), LDL-C, high-density lipoprotein cholesterol (HDL-C) and triglycerides (TG) in consecutive patients with FH before PCSK9-I treatment and after 12 (T12w) and 36 (T36w) weeks of treatment. We evaluated LDL-C target achievement according to different mutations in LDLR. Eighty FH subjects (mean age:54±13.3 years), 39 heterozygous (He) with defective LDLR gene mutations, 30 He with null mutations and 11 compound-He or homozygotes (Ho) were recruited. At baseline, 69 subjects were under maximal lipid lowering therapy (MLLT) and 11 subjects had statin-intolerance. From baseline to T36w we observed an overall 51% reduction in LDL-C. We found no difference in LDL-C changes between subjects with He-defective mutation and He-null mutations both at T12w (p=1.00) and T36w (p=0.538). At T36w, LDL-C target was achieved in 59% of He-defective mutations subjects and in 36% of He-null mutations subgroup (p=0.069), whereas none of compound-He/Ho-FH achieved LDL-C target. Conclusions After 36 weeks there were no differences in response to PCSK9-I therapy between different groups of He-FH subjects. Response to PCSK9-I was significantly lower in carriers of compound-He/Ho mutations. Registration number for clinical trials: NCT04313270 extension.

Familial Hypercholesterolemia (FH) is the most common genetic disease caused by variants in LDLR, APOB, PCSK9 genes; it is characterized by high levels of LDL-cholesterol and premature cardiovascular disease. We aim to perform a retrospective analysis of a genetically screened population (528 unrelated patients - 342 adults and 186 children) to evaluate the biochemical and clinical correlations with the different genetic statuses. Genetic screening was performed by traditional sequencing and some patients were re-analyzed by next-generation-sequencing. Pathogenic variants, mainly missense in the LDLR gene, were identified in 402/528 patients (76.1%), including 4 homozygotes, 17 compound heterozygotes and 1 double heterozygotes. A gradual increase of LDL-cholesterol was observed from patients without pathogenic variants to patients with a defective variant, to patients with a null variant and to patients with two variants. Six variants accounted for 51% of patients; a large variability of LDL-cholesterol was observed among patients carrying the same variant. The frequency of pathogenic variants gradually increased from unlikely FH to definite FH, according to the Dutch Lipid Clinic Network criteria. Genetic diagnosis can help prognostic evaluation of FH patients, discriminating between the different genetic statuses or variant types. Clinical suspicion of FH should be considered even if few symptoms are present or if LDL-cholesterol is only mildly increased. This article is protected by copyright. All rights reserved.

Summary Systematic insight into cellular dysfunction can improve understanding of disease etiology, risk assessment, and patient stratification. We present a multiparametric high-content imaging platform enabling quantification of low-density lipoprotein (LDL) uptake and lipid storage in cytoplasmic droplets of primary leukocyte subpopulations. We validate this platform with samples from 65 individuals with variable blood LDL-cholesterol (LDL-c) levels, including familial hypercholesterolemia (FH) and non-FH subjects. We integrate lipid storage data into another readout parameter, lipid mobilization, measuring the efficiency with which cells deplete lipid reservoirs. Lipid mobilization correlates positively with LDL uptake and negatively with hypercholesterolemia and age, improving differentiation of individuals with normal and elevated LDL-c. Moreover, combination of cell-based readouts with a polygenic risk score for LDL-c explains hypercholesterolemia better than the genetic risk score alone. This platform provides functional insights into cellular lipid trafficking and has broad possible applications in dissecting the cellular basis of metabolic disorders.

Background and aims Familial hypercholesterolemia (FH) is a monogenic disease characterized by high levels of low-density lipoprotein cholesterol and premature atherosclerotic cardiovascular disease. FH is caused by loss of function mutations in genes encoding LDL receptor (LDLR), and Apolipoprotein B (APOB) or gain of function (GOF) mutations in proprotein convertase subtilisin/kexin type 9 (PCSK9). In this study, we identified a novel variant in PCSK9, p.(Arg499His), located in the C-terminal domain, in two unrelated FH patients from Spain and Italy. Methods We studied familial segregation and determined variant activity in vitro. Results We determined PCSK9 expression, secretion and activity of the variant in transfected HEK293 cells; extracellular activity of the recombinant p.(Arg499His) PCSK9 variant in HEK 293 and HepG2 cells; PCSK9 affinity to the LDL receptor at neutral and acidic pH; the mechanism of action of the p.(Arg499His) PCSK9 variant by co-transfection with a soluble construct of the LDL receptor and by determining total PCSK9 intracellular accumulation when endosomal acidification is impaired and when an excess of soluble LDLr is present in the culture medium. Our results show high LDL-C concentrations and FH phenotype in p.(Arg499His) carriers. In vitro functional characterization shows that p.(Arg499His) PCSK9 variant causes a reduction in LDLr expression and LDL uptake. An intracellular activity for this variant is also shown when blocking the activity of secreted PCSK9 and by inhibiting endosomal acidification. Conclusions We demonstrated that p.(Arg499His) PCSK9 variant causes a direct intracellular degradation of LDLr therefore causing FH by reducing LDLr availability.

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Background and aim: Protein convertase subtilisin kexin type 9 (PCSK-9) inhibitors demonstrated efficacy in cholesterol reduction and in the prevention of cardiovascular events. We evaluated changes in lipid profile and carotid stiffness in patients with familial hypercholesterolemia during 12 weeks of treatment with a PCSK-9 inhibitor, Evolocumab®. Methods and results: Patients with familial hypercholesterolemia starting a treatment with Evolocumab® were included. Total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), small dense LDL (assessed by LDL score) and carotid stiffness were evaluated before starting treatment with Evolocumab® and during 12 weeks of treatment. Twenty-five subjects were enrolled (52% males, mean age 51.5 years). TC and LDL-C were reduced of 38% and 52%, respectively during treatment, with LDL score reduced of 46.1%. In parallel, carotid stiffness changed from 8.8 (IQR: 7.0–10.4) m/sec to 6.6 (IQR: 5.4–7.5) m/sec, corresponding to a median change of 21.4% (p < 0.001), with a significant increase in carotid distensibility (from 12.1, IQR: 8.73–19.3 kPA−1 × 10−3 at T0 to 21.8, IQR: 16.6–31.8 kPA−1 × 10−3 at T12w) corresponding to a median change of 62.8% (p < 0.001). A multivariate analysis showed that changes in LDL score were independently associated with changes in carotid stiffness (β = 0.429, p = 0.041). Conclusion: Small dense LDL reduction, as assessed by LDL score, is associated with changes in carotid stiffness in patients with familial hypercholesterolemia treated with Evolocumab®.

Cerebrotendinous Xanthomatosis (CTX) is an autosomal recessive defect of the alternative pathway of bile acid biosynthesis, due to the deficiency of mitochondrial cytochrome P450 sterol 27-hydroxylase enzyme encoded by CYP27A1. The deficit of sterol 27-hydroxylase raises cholestanol in plasma and tissues of affected patients. Although there is a marked variability of signs, symptoms, severity and age of onset, the main clinical manifestations of CTX include chronic diarrhea, bilateral cataract, tendon xanthomas and neurological dysfunction. Herein, we report the clinical, biochemical and molecular characterization of a Caucasian female affected by CTX diagnosed at 28years. The patient’s clinical history revealed neurological and behavioral manifestations already at fifth year of life, following by bilateral cataract and chronic diarrhea without xanthomas. At diagnosis, an involvement of the cervical spinal cord was also observed on MRI. Sterols profile analysis in plasma and red blood cell membranes showed very high cholestanol levels. CYP27A1 sequencing revealed a new variant (e.g., c.850_854delinsCTC) at homozygous status. The follow-up after 5months of chenodeoxycholic acid treatment showed a decrease of plasma cholestanol of 64%. After 1 year, the patient showed normalization of bowel function, reduction of risk of falls, improvement of cognitive function although brain and spine MRI and other instrumental examinations remained unchanged. This case highlights the variability of the CTX phenotype that makes it difficult to reach an early diagnosis. Biochemical and/or molecular screening of CTX should be taken into account to early start the pharmacological treatment limiting neurological damages.

Galectin-3 (Gal-3) is a β-galactoside-binding protein belonging to the lectin family with pleiotropic regulatory activities and several physiological cellular functions, such as cellular growth, proliferation, apoptosis, differentiation, cellular adhesion, and tissue repair. Inflammation, tissue fibrosis and angiogenesis are the main processes in which Gal-3 is involved. It is implicated in the pathogenesis of several diseases, including organ fibrosis, chronic inflammation, cancer, atherosclerosis and other cardiovascular diseases (CVDs). This review aims to explore the connections of Gal-3 with cardiovascular diseases since they represent a major cause of morbidity and mortality. We herein discuss the evidence on the pro-inflammatory role of Gal-3 in the atherogenic process as well as the association with plaque features linked to lesion stability. We report the biological role and molecular mechanisms of Gal-3 in other CVDs, highlighting its involvement in the development of cardiac fibrosis and impaired myocardium remodelling, resulting in heart failure and atrial fibrillation. The role of Gal-3 as a prognostic marker of heart failure is described together with possible diagnostic applications to other CVDs. Finally, we report the tentative use of Gal-3 inhibition as a therapeutic approach to prevent cardiac inflammation and fibrosis.

Aim: Atherosclerosis is one of the leading causes of death and morbidity worldwide. Plaque instability is the most relevant predisposing factor for acute events. We aim to better understand the role of Galectin-3 (Gal-3) and Lipoprotein(a) (Lp(a)) in atherosclerotic process and to define their potential role as peripheral marker of plaque instability. Methods: Advanced human carotid plaques from 99 patients undergoing carotid endarterectomy were classified histologically according to American Heart Association (AHA) guidelines. Plaque complication (type VI) is defined by the presence of hemorrhage, ulceration or hematoma. Gal-3 and Lp(a) was measured in serum samples from patients and 17 healthy controls by immunometric assays. The quantification of Gal-3 in plaque was performed by immunohistochemistry. Results: Gal-3 and Lp(a)/LDL serum levels showed increased levels in patients compared with the control group (p¼0.0001 and p¼0.001 respectively). No correlations have been observed between Gal-3 serum levels and Gal-3 levels in atherosclerotic plaques. No differences were found between Gal-3 serum levels among the different plaques types, nor between complicated and uncomplicated plaques. At multivariate logistic regression the presence of complicated lesion or patient symptoms (surrogate marker of plaque instability) are not related to both Gal-3 or Lp(a)/LDL. Conclusions: Our data showed that Gal-3 and Lp(a) are good markers of advanced atherosclerotic plaque. The absence of differences among the different lesion types suggest that the increase of Gal-3 and Lp(a) is independent by the specific plaque features. Both Gal-3 and Lp(a)/LDL levels cannot be considered markers of plaque instability.

Single nucleotide polymorphisms (SNPs) are the foremost part of many genome association studies. Selecting a subset of SNPs that is sufficiently informative but still small enough to reduce the genotyping overhead is an important step towards disease-gene association. In this work, a Random Forest (RF) approach to informative SNPs selection in Familial Combined Hyperlipidemia (FCH) is proposed. FCH is the most common form of familial hyperlipidemia. Affected patients have elevated levels of plasma triglycerides and/or total cholesterol and show increased risk of premature coronary heart disease. In order to identify susceptibility markers for FCH we perform the analysis of 21 SNPs in ten genes associated with high cardiovascular risk. RF appears to be a useful technique in identifying gene polymorphisms involved in FCH: the identified SNPs confirmed some variants in a couple of genes as genetic markers of FCH as proved in various studies in scientific literature and lead us to report for the first time a further gene association with FCH. This result could be promising and encourages to further investigate on the role of the identified gene in the development of FCH phenotype.

BACKGROUND: Atherosclerosis, a multi-factorial disease, is the main determinant of cardiovascular disease (CVD) leading to high mortality and morbidity in westernized countries. Gene polymorphisms and gene expression related to the atherosclerosis process can be identified using a genome-wide approach or looking for candidate disease-causing genes. METHODS: Using genome wide strategy or candidate gene approach various genes, including paraoxonase genes, which are involved in lipid metabolism, oxidation, inflammation and coagulation, have been associated to atherosclerosis. CONCLUSION: Evaluation of gene polymorphisms, together with traditional and novel biochemical parameters, may help identify individuals at a high risk for CVD. Genetic characterization of susceptibility genes may also lead to new drugs for atherosclerosis prevention and treatment. In addition, gene expression studies can provide novel insights into the molecular mechanism of lesion development and progression.