Publications by authors named "Ilaria Zanotti"

44 Publications

HDL and reverse cholesterol transport in humans and animals: Lessons from pre-clinical models and clinical studies.

Biochim Biophys Acta Mol Cell Biol Lipids 2021 Oct 9;1867(1):159065. Epub 2021 Oct 9.

Division of Translational Medicine & Human Genetics, Perelman School of Medicine at the University of Pennsylvania, 3600 Spruce Street, Philadelphia, PA 19104, USA.

The ability to accept cholesterol from cells and to promote reverse cholesterol transport (RCT) represents the best characterized antiatherogenic function of HDL. Studies carried out in animal models have unraveled the multiple mechanisms by which these lipoproteins drive cholesterol efflux from macrophages and cholesterol uptake to the liver. Moreover, the influence of HDL composition and the role of lipid transporters have been clarified by using suitable transgenic models or through experimental design employing pharmacological or nutritional interventions. Cholesterol efflux capacity (CEC), an in vitro assay developed to offer a measure of the first step of RCT, has been shown to associate with cardiovascular risk in several human cohorts, supporting the atheroprotective role of RCT in humans as well. However, negative data in other cohorts have raised concerns on the validity of this biomarker. In this review we will present the most relevant data documenting the role of HDL in RCT, as assessed in classical or innovative methodological approaches.
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http://dx.doi.org/10.1016/j.bbalip.2021.159065DOI Listing
October 2021

Impact of Dietary Lipids on the Reverse Cholesterol Transport: What We Learned from Animal Studies.

Nutrients 2021 Jul 30;13(8). Epub 2021 Jul 30.

Dipartimento di Scienze degli Alimenti e del Farmaco, Università di Parma, Parco Area delle Scienze 27/A, 43124 Parma, Italy.

Reverse cholesterol transport (RCT) is a physiological mechanism protecting cells from an excessive accumulation of cholesterol. When this process begins in vascular macrophages, it acquires antiatherogenic properties, as has been widely demonstrated in animal models. Dietary lipids, despite representing a fundamental source of energy and exerting multiple biological functions, may induce detrimental effects on cardiovascular health. In the present review we summarize the current knowledge on the mechanisms of action of the most relevant classes of dietary lipids, such as fatty acids, sterols and liposoluble vitamins, with effects on different steps of RCT. We also provide a critical analysis of data obtained from experimental models which can serve as a valuable tool to clarify the effects of dietary lipids on cardiovascular disease.
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http://dx.doi.org/10.3390/nu13082643DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8401548PMC
July 2021

Dysfunctional High-Density Lipoproteins in Type 2 Diabetes Mellitus: Molecular Mechanisms and Therapeutic Implications.

J Clin Med 2021 May 21;10(11). Epub 2021 May 21.

Atherosclerosis and Vascular Biology Laboratory (AtheroLab), Cardiology Department, State University of Campinas (Unicamp), Campinas 13084-971, Brazil.

High density lipoproteins (HDLs) are commonly known for their anti-atherogenic properties that include functions such as the promotion of cholesterol efflux and reverse cholesterol transport, as well as antioxidant and anti-inflammatory activities. However, because of some chronic inflammatory diseases, such as type 2 diabetes mellitus (T2DM), significant changes occur in HDLs in terms of both structure and composition. These alterations lead to the loss of HDLs' physiological functions, to transformation into dysfunctional lipoproteins, and to increased risk of cardiovascular disease (CVD). In this review, we describe the main HDL structural/functional alterations observed in T2DM and the molecular mechanisms involved in these T2DM-derived modifications. Finally, the main available therapeutic interventions targeting HDL in diabetes are discussed.
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http://dx.doi.org/10.3390/jcm10112233DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8196572PMC
May 2021

Rationale and design of the expanded combination of evolocumab plus empagliflozin in diabetes: EXCEED-BHS3 trial.

Ther Adv Chronic Dis 2020 28;11:2040622320959248. Epub 2020 Sep 28.

Brazilian Heart Study Group, Cardiology Division, State University of Campinas, Rua Tessália Vieira de Camargo, 126., Cidade Universitária Zeferino Vaz, Campinas, São Paulo, 13084-971, Brazil.

Background: Patients with type 2 diabetes mellitus (T2DM) remain at increased cardiovascular residual risk and endothelial dysfunction, even after optimizing metabolic control and treatment by sodium-glucose-2 transporter inhibitors (SGLT2-is). The present study was based on the hypothesis that proprotein convertase subtilisin/kexin 9 inhibitor (PCSK9i) therapy may mitigate endothelial dysfunction in T2DM patients who are on regular treatment by SGLT2-i.

Methods: The EXCEED-BHS3 is a prospective, single-center, investigator-blinded, open-label, randomized clinical trial. Participants ( = 110) will be randomized (1:1) to either empagliflozin 25 mg/day alone or empagliflozin 25 mg/day plus evolocumab 140 mg every 2 weeks in addition to optimal medical care. The primary endpoint was defined as the change in the 1-min flow-mediated dilation (FMD) after 16 weeks of treatment. The secondary endpoint is the FMD change after ischemia/reperfusion injury protocol (reserve FMD) after 16 weeks of treatment. Exploratory outcomes comprise the change in FMD and reserve FMD after 8 weeks of treatment and the change after 16 weeks of treatment in the following parameters: plasma levels of nitric oxide, vascular cell adhesion molecule-1 and isoprostane, high-density lipoprotein (HDL) and low-density lipoprotein subfractions profile, HDL function, blood pressure, body mass index, waist circumference and adipokines.

Conclusion: This will be the first study to evaluate the add-on effect of PCSK9i on endothelial function of T2DM patients under regular use of empagliflozin.

Trial Registration: ClinicalTrials.gov identifier: NCT03932721.
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http://dx.doi.org/10.1177/2040622320959248DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7534094PMC
September 2020

The adaptation of lipid profile of human fibroblasts to alginate 2D films and 3D printed scaffolds.

Biochim Biophys Acta Gen Subj 2021 01 18;1865(1):129734. Epub 2020 Sep 18.

Department of Food and Drug Science, University of Parma, Parco Area delle Scienze 27/A, 43124 Parma, Italy. Electronic address:

Background: The investigation of the interactions between cells and active materials is pivotal in the emerging 3D printing-biomaterial application fields. Here, lipidomics has been used to explore the early impact of alginate (ALG) hydrogel architecture (2D films or 3D printed scaffolds) and the type of gelling agent (CaCl or FeCl) on the lipid profile of human fibroblasts.

Methods: 2D and 3D ALG scaffolds were prepared and characterized in terms of water content, swelling, mechanical resistance and morphology before human fibroblast seeding (8 days). Using a liquid chromatography-triple quadrupole-tandem mass spectrometry approach, selected ceramides (CER), lysophosphatidylcholines (LPC), lysophosphatidic acids (LPA) and free fatty acids (FFA) were analyzed.

Results: The results showed a clear alteration in the CER expression profile depending of both the geometry and the gelling agent used to prepare the hydrogels. As for LPCs, the main parameter affecting their distribution is the scaffold architecture with a significant decrease in the relative expression levels of the species with higher chain length (C20 to C22) for 3D scaffolds compared to 2D films. In the case of FFAs and LPAs only slight differences were observed as a function of scaffold geometry or gelling agent.

Conclusions: Variations in the cell membrane lipid profile were observed for 3D cell cultures compared to 2D and these data are consistent with activation processes occurring through the mutual interactions between fibroblasts and ALG support. These unknown physiologically relevant changes add insights into the discussion about the relationship between biomaterial and the variations of cell biological functions.
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http://dx.doi.org/10.1016/j.bbagen.2020.129734DOI Listing
January 2021

Three-Dimensional (3D) Printed Silver Nanoparticles/Alginate/Nanocrystalline Cellulose Hydrogels: Study of the Antimicrobial and Cytotoxicity Efficacy.

Nanomaterials (Basel) 2020 Apr 28;10(5). Epub 2020 Apr 28.

Food and Drug Department, University of Parma, Parco Area delle Scienze 27/a, 43124 Parma, Italy.

Here, a formulation of silver nanoparticles (AgNPs) and two natural polymers such as alginate (ALG) and nanocrystalline cellulose (CNC) was developed for the 3D printing of scaffolds with large surface area, improved mechanical resistance and sustained capabilities to promote antimicrobial and cytotoxic effects. Mechanical resistance, water content, morphological characterization and silver distribution of the scaffolds were provided. As for applications, a comparable antimicrobial potency against and was demonstrated by in vitro tests as function of the AgNP concentration in the scaffold (Minimal Inhibitory Concentration value: 10 mg/mL). By reusing the 3D system the antimicrobial efficacy was demonstrated over at least three applications. The cytotoxicity effects caused by administration of AgNPs to hepatocellular carcinoma (HepG2) cell culture through ALG and ALG/CNC scaffold were discussed as a function of time and dose. Finally, the liquid chromatography-mass spectrometry (LC-MS) technique was used for targeted analysis of pro-apoptotic initiation and executioner caspases, anti-apoptotic and proliferative proteins and the hepatocyte growth factor, and provided insights about molecular mechanisms involved in cell death induction.
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http://dx.doi.org/10.3390/nano10050844DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7711489PMC
April 2020

Ephrin or not? Six tough questions on Eph targeting.

Expert Opin Ther Targets 2020 05 26;24(5):403-415. Epub 2020 Mar 26.

Dipartimento di Scienze degli Alimenti e del Farmaco, Università di Parma, Parma, Italy.

: The Eph-ephrin is a cell-cell communication system generating a forward signal in cell expressing Eph receptors and a reverse signal in ephrin-ligand expressing cells. While clearly involved in the insurgence and progression of cancer, the understanding of the molecular mechanisms regulated by this system needs development; this is a hurdle to the development of therapeutic strategies that can target the Eph receptors and/or their ephrin ligands.: We have taken the opportunity to share some key questions on the most effective strategies to target the Eph-ephrin system. This article is based on our experience of the field and therefore is a Perspective and not comprehensive examination of the literature.: Targeting of the Eph-ephrin system has emerged as a potentially valuable approach for cancer therapy. Pharmacological tools have been reported in the last 15 years and these include forward signaling blockers such as kinases inhibitors and antagonists of forward and reverse signaling. Also, biologics including antibodies and recombinant proteins have been developed and some have reached early clinical stages. Data deem the Eph-ephrin system as a signaling axis that is an elusive target. A better understanding of the basic pharmacology behind the activity of available agents and a comprehensive knowledge of the ephrin biology are necessary. We are looking forward to knowing the opinion of the readers.
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http://dx.doi.org/10.1080/14728222.2020.1745187DOI Listing
May 2020

Excess weight mediates changes in HDL pool that reduce cholesterol efflux capacity and increase antioxidant activity.

Nutr Metab Cardiovasc Dis 2020 02 23;30(2):254-264. Epub 2019 Sep 23.

Laboratory of Atherosclerosis and Vascular Biology, Faculty of Medical Sciences, State University of Campinas, São Paulo, Brazil; Department of Internal Medicine, Cardiology Division, Faculty of Medical Sciences, State University of Campinas, São Paulo, Brazil. Electronic address:

Background And Aim: Obesity-related decline in high-density lipoprotein (HDL) functions such as cholesterol efflux capacity (CEC) has supported the notion that this lipoprotein dysfunction may contribute for atherogenesis among obese patients. We investigated if potentially other HDL protective actions may be affected with weight gain and these changes may occur even before the obesity range in a cross-sectional analysis.

Methods And Results: Lipid profile, body mass index (BMI), biochemical measurements, and carotid intima-media thickness (cIMT) were obtained in this cross-sectional study with 899 asymptomatic individuals. Lipoproteins were separated by ultracentrifugation and HDL physical-chemical characterization, CEC, antioxidant activity, anti-inflammatory activity, HDL-mediated platelet aggregation inhibition were measured in a randomly-selected subgroup (n = 101). Individuals with increased HDL-C had an attenuated increase in cIMT with elevation of BMI (interaction effect β = -0.054; CI 95% -0.0815, -0.0301). CEC, HDL-C, HDL size and HDL-antioxidant activity were negatively associated with cIMT. BMI was inversely correlated with HDL-mediated inhibition of platelet aggregation (Spearman's rho -0.157, p < 0.03) and CEC (Spearman's rho -0.32, p < 0.001), but surprisingly it was directly correlated with the antioxidant activity (Spearman's rho 0.194, p = 0.052). Thus, even in non-obese, non-diabetic individuals, increased BMI is associated with a wide change in protective functions of HDL, reducing CEC and increasing antioxidant activity. In these subjects, decreased HDL concentration, size or function are related to increased atherosclerotic burden.

Conclusion: Our findings demonstrate that in non-obese, non-diabetic individuals, the increasing values of BMI are associated with impaired protective functions of HDL and concomitant increase in atherosclerotic burden.
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http://dx.doi.org/10.1016/j.numecd.2019.09.017DOI Listing
February 2020

Lipid trafficking in cardiovascular disease.

Adv Clin Chem 2019 29;92:105-140. Epub 2019 May 29.

Department of Food and Drug, University of Parma, Parma, Italy.

The reduction of plasma apolipoprotein B (apoB) containing lipoproteins has long been pursued as the main modifiable risk factor for the development of cardiovascular disease (CVD). This has led to an intense search for strategies aiming at reducing plasma apoB-lipoproteins, culminating in reduction of overall CV risk. Despite 3 decades of progress, CVD remains the leading cause of morbidity and mortality worldwide and, as such, new therapeutic targets are still warranted. Clinical and preclinical research has moved forward from the original concept, under which some lipids must be accumulated and other removed to achieve the ideal condition in disease prevention, into the concept that mechanisms that orchestrate lipid movement between lipoproteins, cells and organelles is equally involved in CVD. As such, this review scrutinizes potentially atherogenic changes in lipid trafficking and assesses the molecular mechanisms behind it. New developments in risk assessment and new targets for the mitigation of residual CVD risk are also addressed.
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http://dx.doi.org/10.1016/bs.acc.2019.04.002DOI Listing
October 2019

Reciprocal Multifaceted Interaction Between HDL (High-Density Lipoprotein) and Myocardial Infarction.

Arterioscler Thromb Vasc Biol 2019 08 13;39(8):1550-1564. Epub 2019 Jun 13.

Lipoprotein Metabolism Section, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD (L.S., A.T.R.).

Despite decades of therapeutic advances, myocardial infarction remains a leading cause of death worldwide. Recent studies have identified HDLs (high-density lipoproteins) as a potential candidate for mitigating coronary ischemia/reperfusion injury via a broad spectrum of signaling pathways. HDL ligands, such as S1P (sphingosine-1-phosphate), Apo (apolipoprotein) A-I, clusterin, and miRNA, may influence the opening of the mitochondrial channel, insulin sensitivity, and production of vascular autacoids, such as NO, prostacyclin, and endothelin-1. In parallel, antioxidant activity and sequestration of oxidized molecules provided by HDL can attenuate the oxidative stress that triggers ischemia/reperfusion. Nevertheless, during myocardial infarction, oxidation and the capture of oxidized and proinflammatory molecules generate large phenotypic and functional changes in HDL, potentially limiting its beneficial properties. In this review, new findings from cellular and animal models, as well as from clinical studies, will be discussed to describe the cardioprotective benefits of HDL on myocardial infarction. Furthermore, mechanisms by which HDL modulates cardiac function and potential strategies to mitigate postmyocardial infarction risk damage by HDL will be detailed throughout the review.
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http://dx.doi.org/10.1161/ATVBAHA.119.312880DOI Listing
August 2019

HDL-Targeted Therapies During Myocardial Infarction.

Cardiovasc Drugs Ther 2019 06;33(3):371-381

National Heart, Lung and Blood Institute, National Institutes of Health, 10 Center Dr. Building 10 8N222, Bethesda, MD, USA.

It is now apparent that a variety of deleterious mechanisms intrinsic to myocardial infarction (MI) exists and underlies its high residual lethality. Indeed, despite effective coronary patency therapies, ischemia and reperfusion (I/R) injury accounts for about 50% of the infarcted mass. In this context, recent studies in animal models have demonstrated that coronary reperfusion with high-density lipoproteins (HDL) may reduce MI size in up to 30%. A spectrum of mechanisms mediated by either HDL-related apolipoproteins or phospholipids attenuates myocardial cell death. Hence, promising therapeutic approaches such as infusion of reconstituted HDL particles, new HDL by genomic therapy, or the infusion of apoA-I mimetic peptides have been sought as a way of ensuring protection against I/R injury. In this review, we will explore the limitations and potential therapeutic effects of HDL therapies during the acute phase of MI.
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http://dx.doi.org/10.1007/s10557-019-06865-1DOI Listing
June 2019

Polyphenol Health Effects on Cardiovascular and Neurodegenerative Disorders: A Review and Meta-Analysis.

Int J Mol Sci 2019 Jan 16;20(2). Epub 2019 Jan 16.

Dipartimento di Scienze degli Alimenti e del Farmaco, Università di Parma, Parco Area delle Scienze 27/A, 43124 Parma, Italy.

Several studies have demonstrated that polyphenol-enriched diets may have beneficial effects against the development of degenerative diseases, including atherosclerosis and disorders affecting the central nervous system. This activity has been associated not only with antioxidant and anti-inflammatory properties, but also with additional mechanisms, such as the modulation of lipid metabolism and gut microbiota function. However, long-term studies on humans provided controversial results, making the prediction of polyphenol impact on health uncertain. The aim of this review is to provide an overview and critical analysis of the literature related to the effects of the principal dietary polyphenols on cardiovascular and neurodegenerative disorders. We critically considered and meta-analyzed randomized controlled clinical trials involving subjects taking polyphenol-based supplements. Although some polyphenols might improve specific markers of cardiovascular risk and cognitive status, many inconsistent data are present in literature. Therefore, definitive recommendations for the use of these compounds in the prevention of cardiovascular disease and cognitive decline are currently not applicable. Once pivotal aspects for the definition of polyphenol bioactivity, such as the characterization of pharmacokinetics and safety, are addressed, it will be possible to have a clear picture of the realistic potential of polyphenols for disease prevention.
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http://dx.doi.org/10.3390/ijms20020351DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6359281PMC
January 2019

Alcohol Pattern Consumption Differently Affects the Efficiency of Macrophage Reverse Cholesterol Transport in Vivo.

Nutrients 2018 Dec 3;10(12). Epub 2018 Dec 3.

Dipartimento di Scienze degli Alimenti e del Farmaco, Università di Parma, Parco Area delle Scienze 27/A, 43124 Parma, Italy.

It has been well established that moderate alcohol consumption inversely correlates with cardiovascular morbidity and mortality, whereas binge alcohol drinking increases cardiovascular disease risk. The aim of this study was to assess in vivo the impact of different drinking patterns on reverse cholesterol transport (RCT); the atheroprotective process leading to the removal of excess cholesterol from the body. RCT was measured with a standardized, radioisotope-based technique in three groups of atherosclerosis-prone apolipoprotein E knock out mice: Placebo group, receiving water, which would mimic the abstainers; moderate group, receiving 0.8 g/kg alcohol/day for 28 days, which would mimic a moderate intake; binge group, receiving 0.8 g/kg alcohol/day for 5 days/week, followed by the administration of 2.8 g/kg alcohol/day for 2 days/week, which would mimic a heavy intake in a short period. Mice in the binge drinking group displayed an increase in total cholesterol, high density lipoprotein cholesterol (HDL-c) and non-HDL-c (all < 0.0001 vs. placebo), and a significantly reduced elimination of fecal cholesterol. The moderate consumption did not lead to any changes in circulating lipids, but slightly improved cholesterol mobilization along the RCT pathway. Overall, our data confirm the importance of considering not only the total amount, but also the different consumption patterns to define the impact of alcohol on cardiovascular risk.
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http://dx.doi.org/10.3390/nu10121885DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6316025PMC
December 2018

Anti-Atherosclerotic Effect of a Polyphenol-Rich Ingredient, Oleactiv, in a Hypercholesterolemia-Induced Hamster Model.

Nutrients 2018 Oct 15;10(10). Epub 2018 Oct 15.

Dipartimento di Scienze degli Alimenti e del Farmaco, Università degli Studi di Parma, 43126 Parma, Italy.

The development of nutraceutical ingredients has risen as a nutritional solution for health prevention. This study evaluated the effects of Oleactiv, an ingredient developed for the prevention of atherogenesis, in hypercholesterolemic hamsters. Oleactiv is a polyphenol-rich ingredient obtained from artichoke, olive and grape extracts as part of fruit and vegetables commonly consumed within the Mediterranean diet. A total of 21 hamsters were divided into three groups. The standard group (STD) was fed a normolipidemic diet for 12 weeks, while the control group (CTRL) and Oleactiv goup (OLE) were fed a high-fat diet. After sacrifice, the aortic fatty streak area (AFSA), plasmatic total cholesterol (TC), high-density lipoproteins (HDL-C), non-HDL-C and triglycerides (TG), were assessed. The cholesterol efflux capacity (CEC) of hamster plasma was quantified using a radiolabeled technique in murine macrophages J774. OLE administration induced a significant reduction of AFSA (-69%, < 0.0001). Hamsters of the OLE group showed a significant decrease of both non-HDL-C (-173 mmol/L, < 0.05) and TG (-154 mmol/L, < 0.05). Interestingly, OLE induced a significant increase of total CEC (+17,33%, < 0,05). Oleactiv supplementation prevented atheroma development and had positive effects on the lipid profile of hypercholesterolemic hamsters. The increased CEC underlines the anti-atherosclerotic mechanism at the root of the atheroma reduction observed.
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http://dx.doi.org/10.3390/nu10101511DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6213376PMC
October 2018

Cholesterol efflux capacity does not associate with coronary calcium, plaque vulnerability, and telomere length in healthy octogenarians.

J Lipid Res 2018 04 7;59(4):714-721. Epub 2018 Feb 7.

Department of Food and Drug, University of Parma, Parma, Italy. Electronic address:

Several studies have revealed that traditional risk factors are less effective in predicting CVD risk in the elderly, suggesting the need to identify new biomarkers. Here, we evaluated the association between serum cholesterol efflux capacity (CEC), an atheroprotective property of HDL recently identified as a novel marker of CVD risk, and atherosclerotic burden in a cohort of very old, healthy individuals. Serum CEC values were not significantly correlated either with calcium score or with markers of vulnerable plaque, such as positive remodeling, hypodensity, spotty calcification, or napking-ring sign. In addition, no association was detected between CEC and telomere length, a marker of biological aging that has been linked to atherosclerosis extent. Interestingly, elderly subjects presented a remarkably higher CEC (+30.2%; < 0.0001) compared with values obtained from a cohort of sex-matched, cardiovascular event-free, middle-aged individuals. In conclusion, serum CEC is not related to traditional risk factors in very old, cardiovascular event-free subjects, but has significantly higher values compared with a healthy, younger population. Whether this improved HDL functionality may represent a protective factor in CVD onset must be established in future studies.
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http://dx.doi.org/10.1194/jlr.P079525DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5880496PMC
April 2018

PCSK9 induces a pro-inflammatory response in macrophages.

Sci Rep 2018 02 2;8(1):2267. Epub 2018 Feb 2.

Dipartimento di Scienze del Farmaco, Università degli Studi di Padova, Padua, Italy.

Intraplaque release of inflammatory cytokines from macrophages is implicated in atherogenesis by inducing the proliferation and migration of media smooth muscle cells (SMCs). PCSK9 is present and released by SMCs within the atherosclerotic plaque but its function is still unknown. In the present study, we tested the hypothesis that PCSK9 could elicit a pro-inflammatory effect on macrophages. THP-1-derived macrophages and human primary macrophages were exposed to different concentrations (0.250 ÷ 2.5 µg/ml) of human recombinant PCSK9 (hPCSK9). After 24 h incubation with 2.5 µg/ml PCSK9, a significant induction of IL-1β, IL-6, TNF-α, CXCL2, and MCP1 mRNA, were observed in both cell types. Co-culture of THP-1 macrophages with HepG2 overexpressing hPCSK9 also showed the induction of TNF-α (2.4 ± 0.5 fold) and IL-1β (8.6 ± 1.8 fold) mRNA in macrophages. The effect of hPCSK9 on TNF-α mRNA in murine LDLR bone marrow macrophages (BMM) was significantly impaired as compared to wild-type BMM (4.3 ± 1.6 fold vs 31.1 ± 6.1 fold for LDLR and LDLR, respectively). Finally, a positive correlation between PCSK9 and TNF-α plasma levels of healthy adult subjects (males 533, females 537) was observed (B = 8.73, 95%CI 7.54 ÷ 9.93, p < 0.001). Taken together, the present study provides evidence of a pro-inflammatory action of PCSK9 on macrophages, mainly dependent by the LDLR.
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http://dx.doi.org/10.1038/s41598-018-20425-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5797178PMC
February 2018

The Gut Microbial Metabolite Trimethylamine-N-Oxide Is Present in Human Cerebrospinal Fluid.

Nutrients 2017 Sep 22;9(10). Epub 2017 Sep 22.

Dipartimento di Scienze degli Alimenti e del Farmaco, Università degli Studi di Parma, Parco Area delle Scienze 27/A, 43124 Parma, Italy.

Trimethylamine--oxide (TMAO) is a small organic molecule, derived from the intestinal and hepatic metabolism of dietary choline and carnitine. Although the involvement of TMAO in the framework of many chronic diseases has been recently described, no evidence on its putative role in the central nervous system has been provided. The aim of this study was to evaluate whether TMAO is present at detectable levels in human cerebrospinal fluid (CSF). CSF was collected for diagnostic purposes from 58 subjects by lumbar puncture and TMAO was quantified by using liquid chromatography coupled with multiple-reaction monitoring mass spectrometry. The molecule was detected in all samples, at concentrations ranging between 0.11 and 6.43 µmol/L. Further analysis on CSF revealed that a total of 22 subjects were affected by Alzheimer's disease (AD), 16 were affected by non-AD related dementia, and 20 were affected by other neurological disorders. However, the stratification of TMAO levels according to the neurological diagnoses revealed no differences among the three groups. In conclusion, we provide the first evidence that TMAO can be assessed in human CSF, but the actual impact of this dietary metabolite in the patho-physiolgy of the central nervous system requires further study.
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http://dx.doi.org/10.3390/nu9101053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5691670PMC
September 2017

Targeting Eph/ephrin system in cancer therapy.

Eur J Med Chem 2017 Dec 18;142:152-162. Epub 2017 Jul 18.

Department of Food and Drug, University of Parma, Viale delle Scienze 27/a, 43124 Parma, Italy. Electronic address:

It is well established that the Eph/ephrin system plays a central role in the embryonic development, with minor implications in the physiology of the adult. However, it is overexpressed and deregulated in a variety of tumors, with a primary involvement in tumorigenesis, tumor angiogenesis, metastasis development, and cancer stem cell regeneration. Targeting the Eph/ephrin system with biologicals, including antibodies and recombinant proteins, reduces tumor growth in animal models of hematological malignancies, breast, prostate, colon, head and neck cancers and glioblastoma. Currently, some of these biopharmaceutical agents are under investigations in phase I or phase II clinical trials. Peptides and small molecules targeting protein-protein-interaction (PPI) are in the late preclinical phase where they are showing promising activity in models of glioblastoma, ovarian and lung cancer. The present review summarizes the most critical findings proposing the Eph/ephrin signaling system as a new target in molecularly targeted oncology.
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http://dx.doi.org/10.1016/j.ejmech.2017.07.029DOI Listing
December 2017

Phenyl-γ-valerolactones, flavan-3-ol colonic metabolites, protect brown adipocytes from oxidative stress without affecting their differentiation or function.

Mol Nutr Food Res 2017 09 12;61(9). Epub 2017 Apr 12.

The Laboratory of Phytochemicals in Physiology, Department of Food & Drug, University of Parma, Parma, Italy.

Scope: Consumption of products rich in flavan-3-ols, such as tea and cocoa, has been associated with decreased obesity, partially dependent on their capacity to enhance energy expenditure. Despite these phenolics having been reported to increase the thermogenic program in brown and white adipose tissue, flavan-3-ols are vastly metabolised in vivo to phenyl-γ-valerolactones. Therefore, we hypothesize that phenyl-γ-valerolactones may directly stimulate the differentiation and the activation of brown adipocytes.

Methods And Results: Immortalized brown pre-adipocytes were differentiated in presence of (R)-5-(3',4'-dihydroxyphenyl)-γ-valerolactone (VL1), (R)-5-(3´-hydroxyphenyl)-γ-valerolactone-4'-O-sulphate (VL2), (R)-5-phenyl-γ-valerolactone-3´,4´-di-O-sulphate (VL3), at concentrations of 2 or 10μM, whereas fully differentiated brown adipocyte were treated acutely (6-24h). None of the treatments regulated the expression levels of the uncouple protein 1, nor of the main transcription factors involved in brown adipogenesis. Similarly, mitochondrial content was unchanged after treatments. Moreover these compounds did not display peroxisome proliferator-activated receptor γ-agonist activity, as evaluated by luciferase assay, and did not enhance norepinephrine-stimulated lipolysis in mature adipocytes. However, both VL1 and VL2 prevented oxidative stress caused by H O .

Conclusion: Phenyl-γ-valerolactones and their sulphated forms do not influence brown adipocyte development or function at physiological or supraphysiological doses in vitro, but they are active protecting brown adipocytes from increased reactive oxygen species production.
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http://dx.doi.org/10.1002/mnfr.201700074DOI Listing
September 2017

Metadynamics for Perspective Drug Design: Computationally Driven Synthesis of New Protein-Protein Interaction Inhibitors Targeting the EphA2 Receptor.

J Med Chem 2017 01 5;60(2):787-796. Epub 2017 Jan 5.

Dipartimento di Farmacia, Università degli Studi di Parma , Parco Area delle Scienze 27/A, 43124 Parma, Italy.

Metadynamics (META-D) is emerging as a powerful method for the computation of the multidimensional free-energy surface (FES) describing the protein-ligand binding process. Herein, the FES of unbinding of the antagonist N-(3α-hydroxy-5β-cholan-24-oyl)-l-β-homotryptophan (UniPR129) from its EphA2 receptor was reconstructed by META-D simulations. The characterization of the free-energy minima identified on this FES proposes a binding mode fully consistent with previously reported and new structure-activity relationship data. To validate this binding mode, new N-(3α-hydroxy-5β-cholan-24-oyl)-l-β-homotryptophan derivatives were designed, synthesized, and tested for their ability to displace ephrin-A1 from the EphA2 receptor. Among them, two antagonists, namely compounds 21 and 22, displayed high affinity versus the EphA2 receptor and resulted endowed with better physicochemical and pharmacokinetic properties than the parent compound. These findings highlight the importance of free-energy calculations in drug design, confirming that META-D simulations can be used to successfully design novel bioactive compounds.
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http://dx.doi.org/10.1021/acs.jmedchem.6b01642DOI Listing
January 2017

Inhibitory effect of PCSK9 on Abca1 protein expression and cholesterol efflux in macrophages.

Atherosclerosis 2017 01 16;256:1-6. Epub 2016 Nov 16.

Dipartimento di Scienze del Farmaco, Università degli Studi di Padova, Padova, Italy.

Background And Aims: Proprotein convertase subtilisin/kexin type 9 (PCSK9) may have extra-hepatic effects on cholesterol homeostasis of vascular macrophages. In this study, we aimed to investigate PCSK9 role on the anti-atherogenic process of ATP binding cassette transporter A1 (Abca1)-mediated cholesterol efflux.

Methods: Abca1-mediated cholesterol efflux was evaluated by a radioisotopic technique in mouse peritoneal macrophages (MPM) from wild-type (WT) or LDL receptor knock-out (Ldlr) mice exposed to human recombinant PCSK9, in the presence of liver X receptor/retinoid X receptor (LXR/RXR) ligands or acetylated LDL (AcLDL) to stimulate Abca1 expression. Protein and gene expression was evaluated by Western blot and quantitative real time PCR, respectively.

Results: PCSK9 inhibited Abca1-mediated cholesterol efflux induced by LXR/RXR agonists in WT MPM (-55%, p < 0.05) but not in Ldlr MPM. This effect was fully abrogated by the co-incubation with an anti-PCSK9 antibody. The inhibition of Abca1-dependent efflux induced by PCSK9 was associated with a reduction of Abca1 protein expression only in WT cells. Abca1 gene expression was significantly downregulated by PCSK9 in WT macrophages (-64%, p < 0.001) and, to a lesser extent, in MPM lacking Ldlr (-35%, p < 0.001). The inhibitory effect on Abca1-mediated efflux was also confirmed in AcLDL-treated macrophages. PCSK9 had a marginal or no effect on the expression of the lipid transporters Sr-b1 and Abcg1.

Conclusions: PCSK9 plays a direct role on Abca1-mediated cholesterol efflux through a downregulation of Abca1 gene and Abca1 protein expression. This extrahepatic effect may influence relevant steps in the pathogenesis of atherosclerosis, such as foam cell formation.
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http://dx.doi.org/10.1016/j.atherosclerosis.2016.11.019DOI Listing
January 2017

Increased PCSK9 Cerebrospinal Fluid Concentrations in Alzheimer's Disease.

J Alzheimers Dis 2017 ;55(1):315-320

Dipartimento di Scienze del Farmaco, Università degli Studi di Padova, Padova, Italy.

Background: Alzheimer's disease (AD) has been associated with dysregulation of brain cholesterol trafficking and abnormal production of apolipoprotein E isoform 4 (apoE4). Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a protein present in serum and cerebrospinal fluid (CSF) degrading the low-density lipoprotein receptor (LDLr) and other apoE-binding receptors involved in neuron cholesterol uptake. The role of PCSK9 in AD is controversial.

Objective: We compared PCSK9 levels in CSF of AD patients and non-AD controls and looked at correlations with CSF total apoE and apoE4.

Methods: CSF from AD (n = 30) and from age and sex-matched non-AD patients (n = 30) was collected by lumbar puncture for routine diagnosis. CSF PCSK9, total apoE, and apoE4 levels were measured by ELISA. AD patients showed the typical CSF neurobiomarker pattern (decreased Aβ42 and increased tau and phospho-tau) and impaired cognitive performances, as indicated by the scores of the Mini-Mental State Examination test.

Results: PCSK9 levels in CSF were higher in AD than in non-AD subjects (+1.45 fold; p = 0.0049). CSF total apoE concentrations did not differ between the two groups, while apoE4 levels were higher in AD subjects (+3.34 fold; p = 0.0068). Considering all samples, a significant positive correlation was found between PCSK9 and apoE4 (r = 0.4409; p = 0.0006). PCSK9 levels were higher in APOE ɛ4 carriers, reaching statistical significance in the AD group (+1.45 fold; p = 0.0454).

Conclusion: These results report for the first time an alteration of CSF PCSK9 levels in AD and suggest a pathophysiological link between PCSK9, apoE4, and AD.
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http://dx.doi.org/10.3233/JAD-160411DOI Listing
February 2018

Antiatherogenic effects of ellagic acid and urolithins in vitro.

Arch Biochem Biophys 2016 06 15;599:42-50. Epub 2016 Feb 15.

Human Nutrition Unit, Department of Food Science, University of Parma, Via Volturno 39, 43125 Parma, Italy; The Need for Nutrition Education/Innovation Programme (NNEdPro), University of Cambridge, Cambridge, UK. Electronic address:

Atherosclerosis, one of the leading causes of death worldwide, is characterized by impaired endothelial function and lipid metabolism, among other factors. Ellagitannins are a class of phenolic compounds that may play a role in cardiovascular health. This work aimed to study the potential atheroprotective effects of urolithins, ellagitannin-derived gut microbiota metabolites, on different key factors in atherosclerosis development: the ability of monocytes to adhere to endothelial cells and the uptake and efflux of cholesterol by macrophages. The biotransformations urolithins undergo in peripheral cells were also evaluated. Results indicated that some urolithins and ellagic acid were able to reduce the adhesion of THP-1 monocytes to human umbilical vein endothelial cells (HUVECs) and the secretion of a cellular adhesion molecule (sVCAM-1) and pro-inflammatory cytokine (IL-6). Urolithin C, a combination of urolithins A and B, and ellagic acid also decreased the accumulation of cholesterol in THP-1-derived macrophages, but they were not able to promote cholesterol efflux. The analysis of cell media by UHPLC-ESI-MS(n) indicated urolithins and ellagic underwent extensive metabolism, with sulfate and methyl conjugation. This evidence indicates that atherosclerotic processes may be attenuated by urolithins, but future human intervention trials are required to establish if is translated in vivo.
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http://dx.doi.org/10.1016/j.abb.2016.02.017DOI Listing
June 2016

Δ(5)-Cholenoyl-amino acids as selective and orally available antagonists of the Eph-ephrin system.

Eur J Med Chem 2015 Oct 29;103:312-24. Epub 2015 Aug 29.

Dipartimento di Farmacia, Università degli Studi di Parma, Parco Area delle Scienze 27/A, 43124, Parma, Italy. Electronic address:

The Eph receptor-ephrin system is an emerging target for the development of novel anti-angiogenic therapies. Research programs aimed at developing small-molecule antagonists of the Eph receptors are still in their initial stage as available compounds suffer from pharmacological drawbacks, limiting their application in vitro and in vivo. In the present work, we report the design, synthesis and evaluation of structure-activity relationships of a class of Δ(5)-cholenoyl-amino acid conjugates as Eph-ephrin antagonists. As a major achievement of our exploration, we identified N-(3β-hydroxy-Δ(5)-cholen-24-oyl)-L-tryptophan (UniPR1331) as the first small molecule antagonist of the Eph-ephrin system effective as an anti-angiogenic agent in endothelial cells, bioavailable in mice by the oral route and devoid of biological activity on G protein-coupled and nuclear receptors targeted by bile acid derivatives.
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http://dx.doi.org/10.1016/j.ejmech.2015.08.048DOI Listing
October 2015

Evidence for cholesterol-lowering activity by Bifidobacterium bifidum PRL2010 through gut microbiota modulation.

Appl Microbiol Biotechnol 2015 Aug 12;99(16):6813-29. Epub 2015 Apr 12.

Department of Pharmacy, University of Parma, Parma, Italy.

Bifidobacteria are members of the human gut microbiota, which are known to influence the metabolic abilities of their host. Here, we investigated the capabilities of bifidobacteria to reduce cholesterol levels in synthetic growth media, clearly demonstrating assimilation of this molecule by particular bifidobacterial strains, including Bifidobacterium bifidum PRL2010 (LMG S-28692). The transcriptomic analysis of PRL2010 cells cultivated in the presence of cholesterol revealed a significantly increased transcription level of genes encoding putative transporters and reductases, indicative of specific mechanisms for cholesterol assimilation as well as cholesterol conversion to coprostanol. Cholesterol lowering activity of B. bifidum PRL2010 cells was further evaluated by means of an in vivo murine model, showing that the fecal microbiota of mice is modified toward those bacteria involved in the metabolism of cholesterol.
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http://dx.doi.org/10.1007/s00253-015-6564-7DOI Listing
August 2015

Cholesterol efflux and reverse cholesterol transport.

Handb Exp Pharmacol 2015 ;224:181-206

Department of Pharmacy, University of Parma, Parco Area delle Scienze 27/A, 43124, Parma, Italy.

Both alterations of lipid/lipoprotein metabolism and inflammatory events contribute to the formation of the atherosclerotic plaque, characterized by the accumulation of abnormal amounts of cholesterol and macrophages in the artery wall. Reverse cholesterol transport (RCT) may counteract the pathogenic events leading to the formation and development of atheroma, by promoting the high-density lipoprotein (HDL)-mediated removal of cholesterol from the artery wall. Recent in vivo studies established the inverse relationship between RCT efficiency and atherosclerotic cardiovascular diseases (CVD), thus suggesting that the promotion of this process may represent a novel strategy to reduce atherosclerotic plaque burden and subsequent cardiovascular events. HDL plays a primary role in all stages of RCT: (1) cholesterol efflux, where these lipoproteins remove excess cholesterol from cells; (2) lipoprotein remodeling, where HDL undergo structural modifications with possible impact on their function; and (3) hepatic lipid uptake, where HDL releases cholesterol to the liver, for the final excretion into bile and feces. Although the inverse association between HDL plasma levels and CVD risk has been postulated for years, recently this concept has been challenged by studies reporting that HDL antiatherogenic functions may be independent of their plasma levels. Therefore, assessment of HDL function, evaluated as the capacity to promote cell cholesterol efflux may offer a better prediction of CVD than HDL levels alone. Consistent with this idea, it has been recently demonstrated that the evaluation of serum cholesterol efflux capacity (CEC) is a predictor of atherosclerosis extent in humans.
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http://dx.doi.org/10.1007/978-3-319-09665-0_4DOI Listing
April 2015

Atheroprotective effects of (poly)phenols: a focus on cell cholesterol metabolism.

Food Funct 2015 Jan 4;6(1):13-31. Epub 2014 Nov 4.

Department of Pharmacy, University of Parma, Viale delle Scienze 27/A, 43124 Parma, Italy.

Collated observations from several epidemiological studies have demonstrated that dietary intake of (poly)phenols from nuts, coffee, cocoa, grapes, and berries may protect against the development of atherosclerosis. Whereas this beneficial activity has previously been linked mainly to antioxidant or anti-inflammatory properties, recently emerging data suggest mechanisms by which (poly)phenolic substances can modulate cellular lipid metabolism, thereby mitigating atherosclerotic plaque formation. In this review, both experimental studies and clinical trials investigating the atheroprotective effects of the most relevant dietary (poly)phenols are critically discussed.
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http://dx.doi.org/10.1039/c4fo00670dDOI Listing
January 2015

Combining ligand- and structure-based approaches for the discovery of new inhibitors of the EPHA2-ephrin-A1 interaction.

J Chem Inf Model 2014 Oct 7;54(10):2621-6. Epub 2014 Oct 7.

Dipartimento di Farmacia, Università degli Studi di Parma , Parco Area delle Scienze 27/A, 43124 Parma, Italy.

The EPH receptor A2 (EPHA2) represents an attractive anticancer target. With the aim to identify novel EPHA2 receptor antagonists, a virtual screening campaign, combining shape-similarity and docking calculations, was conducted on a set of commercially available compounds. A combined score, taking into account both ligand- and structure-based results, was then used to identify the most promising candidates. Two compounds, selected among the best-ranked ones, were identified as EPHA2 receptor antagonists with micromolar affinity.
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http://dx.doi.org/10.1021/ci5004619DOI Listing
October 2014
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