Publications by authors named "Gualtiero I Colombo"

50 Publications

Reduction of Cardiac Fibrosis by Interference With YAP-Dependent Transactivation.

Circ Res 2022 Jul 30;131(3):239-257. Epub 2022 Jun 30.

Department of Mechanical and Aerospace Engineering, Politecnico di Torino, Turin, Italy (M.S., D.C., F. Molinari, D.M., U.M.).

Background: Conversion of cardiac stromal cells into myofibroblasts is typically associated with hypoxia conditions, metabolic insults, and/or inflammation, all of which are predisposing factors to cardiac fibrosis and heart failure. We hypothesized that this conversion could be also mediated by response of these cells to mechanical cues through activation of the Hippo transcriptional pathway. The objective of the present study was to assess the role of cellular/nuclear straining forces acting in myofibroblast differentiation of cardiac stromal cells under the control of YAP (yes-associated protein) transcription factor and to validate this finding using a pharmacological agent that interferes with the interactions of the YAP/TAZ (transcriptional coactivator with PDZ-binding motif) complex with their cognate transcription factors TEADs (TEA domain transcription factors), under high-strain and profibrotic stimulation.

Methods: We employed high content imaging, 2-dimensional/3-dimensional culture, atomic force microscopy mapping, and molecular methods to prove the role of cell/nuclear straining in YAP-dependent fibrotic programming in a mouse model of ischemia-dependent cardiac fibrosis and in human-derived primitive cardiac stromal cells. We also tested treatment of cells with Verteporfin, a drug known to prevent the association of the YAP/TAZ complex with their cognate transcription factors TEADs.

Results: Our experiments suggested that pharmacologically targeting the YAP-dependent pathway overrides the profibrotic activation of cardiac stromal cells by mechanical cues in vitro, and that this occurs even in the presence of profibrotic signaling mediated by TGF-β1 (transforming growth factor beta-1). In vivo administration of Verteporfin in mice with permanent cardiac ischemia reduced significantly fibrosis and morphometric remodeling but did not improve cardiac performance.

Conclusions: Our study indicates that preventing molecular translation of mechanical cues in cardiac stromal cells reduces the impact of cardiac maladaptive remodeling with a positive effect on fibrosis.
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http://dx.doi.org/10.1161/CIRCRESAHA.121.319373DOI Listing
July 2022

Whole-Blood Transcriptional Profiles Enable Early Prediction of the Presence of Coronary Atherosclerosis and High-Risk Plaque Features at Coronary CT Angiography.

Biomedicines 2022 Jun 2;10(6). Epub 2022 Jun 2.

Centro Cardiologico Monzino IRCCS, 20138 Milan, Italy.

Existing tools to estimate cardiovascular (CV) risk have sub-optimal predictive capacities. In this setting, non-invasive imaging techniques and omics biomarkers could improve risk-prediction models for CV events. This study aimed to identify gene expression patterns in whole blood that could differentiate patients with severe coronary atherosclerosis from subjects with a complete absence of detectable coronary artery disease and to assess associations of gene expression patterns with plaque features in coronary CT angiography (CCTA). Patients undergoing CCTA for suspected coronary artery disease (CAD) were enrolled. Coronary stenosis was quantified and CCTA plaque features were assessed. The whole-blood transcriptome was analyzed with RNA sequencing. We detected highly significant differences in the circulating transcriptome between patients with high-degree coronary stenosis (≥70%) in the CCTA and subjects with an absence of coronary plaque. Notably, regression analysis revealed expression signatures associated with the Leaman score, the segment involved score, the segment stenosis score, and plaque volume with density <150 HU at CCTA. This pilot study shows that patients with significant coronary stenosis are characterized by whole-blood transcriptome profiles that may discriminate them from patients without CAD. Furthermore, our results suggest that whole-blood transcriptional profiles may predict plaque characteristics.
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http://dx.doi.org/10.3390/biomedicines10061309DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9219643PMC
June 2022

Trained Immunity in Perivascular Adipose Tissue of Abdominal Aortic Aneurysm-A Novel Concept for a Still Elusive Disease.

Front Cell Dev Biol 2022 25;10:886086. Epub 2022 May 25.

Immunology and Functional Genomics Unit, Centro Cardiologico Monzino IRCCS, Milano, Italy.

Abdominal aortic aneurysm (AAA) is a chronic, life-threatening vascular disease whose only therapeutic option is a surgical repair to prevent vessel rupture. The lack of medical therapy results from an inadequate understanding of the etiopathogenesis of AAA. Many studies in animal and human models indicate a 'short-circuiting' of the regulation of the inflammatory-immune response as a major player in the AAA chronic process. In this regard, perivascular adipose tissue (PVAT) has received increasing interest because its dysfunction affects large arteries primarily through immune cell infiltration. Consistently, we have recently produced evidence that innate and adaptive immune cells present in the PVAT of AAAs contribute to sustaining a damaging inflammatory loop. However, it is still unclear how the complex crosstalk between adaptive and innate immunity can be self-sustaining. From our perspective, trained immunity may play a role in this crosstalk. Trained immunity is defined as a form of innate immune memory resulting in enhanced responsiveness to repeated triggers. Specific innate stimuli and epigenetic and metabolic reprogramming events induce and shape trained immunity in myeloid progenitor cells improving host defense, but also contributing to the progression of immune-mediated and chronic inflammatory diseases. Here we present this hypothesis with data from the literature and our observations to support it.
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http://dx.doi.org/10.3389/fcell.2022.886086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9174671PMC
May 2022

Mercaptoalbumin Is Associated with Graft Patency in Patients Undergoing Coronary Artery Bypass Grafting.

Antioxidants (Basel) 2022 Apr 2;11(4). Epub 2022 Apr 2.

Centro Cardiologico Monzino IRCCS, 20138 Milan, Italy.

Coronary artery bypass graft (CABG) surgery still represents the gold standard for patients with complex multivessel coronary artery disease. However, graft occlusion still occurs in a significant proportion of CABG conduits, and oxidative stress is currently considered to be a potential contributor. Human serum albumin (HSA) represents the main antioxidant in plasma through its reduced amino acid Cys34, which can efficiently scavenge several oxidants. In a nested case-control study including 36 patients with occluded grafts and 38 age- and sex-matched patients without occlusion, we assessed the levels of the native mercaptoalbumin (HSA-SH) and oxidized thiolated form of albumin (Thio-HSA) in relation with graft occlusion within 5 years after CABG. We found that the plasma level of preoperative HSA-SH was significantly lower in patients with occluded graft at 5 years follow-up than in patients with graft patency. Furthermore, low HSA-SH remained independently associated with graft occlusion even after adjusting for preoperative D-dimer, a well-known marker of activated coagulation recently found to be associated with graft occlusion. In conclusion, the preoperative level of HSA-SH is independently associated with graft occlusion in CABG and represents a measurable and potentially druggable predictor.
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http://dx.doi.org/10.3390/antiox11040702DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9029960PMC
April 2022

Diagnostic performance of deep learning algorithm for analysis of computed tomography myocardial perfusion.

Eur J Nucl Med Mol Imaging 2022 Jul 23;49(9):3119-3128. Epub 2022 Feb 23.

Centro Cardiologico Monzino, IRCCS, Milan, Italy.

Purpose: To evaluate the diagnostic accuracy of a deep learning (DL) algorithm predicting hemodynamically significant coronary artery disease (CAD) by using a rest dataset of myocardial computed tomography perfusion (CTP) as compared to invasive evaluation.

Methods: One hundred and twelve consecutive symptomatic patients scheduled for clinically indicated invasive coronary angiography (ICA) underwent CCTA plus static stress CTP and ICA with invasive fractional flow reserve (FFR) for stenoses ranging between 30 and 80%. Subsequently, a DL algorithm for the prediction of significant CAD by using the rest dataset (CTP-DL) and stress dataset (CTP-DL) was developed. The diagnostic accuracy for identification of significant CAD using CCTA, CCTA + CTP stress, CCTA + CTP-DL, and CCTA + CTP-DL was measured and compared. The time of analysis for CTP stress, CTP-DL, and CTP-DL was recorded.

Results: Patient-specific sensitivity, specificity, NPV, PPV, accuracy, and area under the curve (AUC) of CCTA alone and CCTA + CTP were 100%, 33%, 100%, 54%, 63%, 67% and 86%, 89%, 89%, 86%, 88%, 87%, respectively. Patient-specific sensitivity, specificity, NPV, PPV, accuracy, and AUC of CCTA + DL and CCTA + DL were 100%, 72%, 100%, 74%, 84%, 96% and 93%, 83%, 94%, 81%, 88%, 98%, respectively. All CCTA + CTP stress, CCTA + CTP-DL, and CCTA + CTP-DL significantly improved detection of hemodynamically significant CAD compared to CCTA alone (p < 0.01). Time of CTP-DL was significantly lower as compared to human analysis (39.2 ± 3.2 vs. 379.6 ± 68.0 s, p < 0.001).

Conclusion: Evaluation of myocardial ischemia using a DL approach on rest CTP datasets is feasible and accurate. This approach may be a useful gatekeeper prior to CTP stress.
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http://dx.doi.org/10.1007/s00259-022-05732-wDOI Listing
July 2022

The α-adrenergic receptor pathway modulating depression influences the risk of arterial thrombosis associated with BDNFVal66Met polymorphism.

Biomed Pharmacother 2022 Feb 26;146:112557. Epub 2021 Dec 26.

Centro Cardiologico Monzino, IRCCS, Milan, Italy. Electronic address:

Depression is associated with thrombotic risk and arterial events, its proper management is strongly recommended in coronary artery disease (CAD) patients. We have previously shown that the Brain-Derived Neurotrophic Factor (BDNF)Val66Met polymorphism, related to depression, is associated with arterial thrombosis in mice, and with an increased risk of acute myocardial infarction in humans. Herein, expanding the previous findings on BDNFVal66Met polymorphism, we show that desipramine, a norepinephrine reuptake-inhibitor, rescues behavioral impairments, reduces the arterial thrombosis risk, abolishes pathological coagulation and platelet hyper-reactivity, normalizes leukocyte, platelet, and bone marrow megakaryocyte number and restores physiological norepinephrine levels in homozygous knock-in BDNF Val66Met (BDNF) mice. The in vitro data confirm the enhanced procoagulant activity and the alpha-adrenergic receptor (α-ADR) overexpression found in BDNF mice and we provide evidence that, in presence of Met variant, norepinephrine is crucial to up-regulate procoagulant activity and to enhance platelet generation. The α-ADR antagonist rauwolscine rescues the prothrombotic phenotype in BDNF mice and reduces procoagulant activity and platelet generation in cells transfected with BDNF plasmid or exposed to pro-BDNF peptide. Finally, we show that homozygous BDNF CAD patients have hyper-reactive platelets overexpressing abundant α-ADR. The great proplatelet release from their megakaryocytes well reflects their higher circulating platelet number compared to BDNF patients. These data reveal an unprecedented described role of Met allele in the dysregulation of norepinephrine/α-ADR pathway that may explain the predisposition to arterial thrombosis. Overall, the development of α-ADR inhibitors might represent a pharmacological treatment for depression-associated thrombotic conditions in this specific subgroup of CAD patients.
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http://dx.doi.org/10.1016/j.biopha.2021.112557DOI Listing
February 2022

Proteomic studies on apoB-containing lipoprotein in cardiovascular research: A comprehensive review.

Mass Spectrom Rev 2021 Nov 8. Epub 2021 Nov 8.

Centro Cardiologico Monzino, IRCCS, Milano, Italy.

The complexity of cardiovascular diseases (CVDs), which remains the leading cause of death worldwide, makes the current clinical pathway for cardiovascular risk assessment unsatisfactory, as there remains a substantial unexplained residual risk. Simultaneous assessment of a large number of plasma proteins may be a promising tool to further refine risk assessment, and lipoprotein-associated proteins have the potential to fill this gap. Technical advances now allow for high-throughput proteomic analysis in a reproducible and cost-effective manner. Proteomics has great potential to identify and quantify hundreds of candidate marker proteins in a sample and allows the translation from isolated lipoproteins to whole plasma, thus providing an individual multiplexed proteomic fingerprint. This narrative review describes the pathophysiological roles of atherogenic apoB-containing lipoproteins and the recent advances in their mass spectrometry-based proteomic characterization and quantitation for better refinement of CVD risk assessment.
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http://dx.doi.org/10.1002/mas.21747DOI Listing
November 2021

Treatment with PCSK9 Inhibitors in Patients with Familial Hypercholesterolemia Lowers Plasma Levels of Platelet-Activating Factor and Its Precursors: A Combined Metabolomic and Lipidomic Approach.

Biomedicines 2021 Aug 23;9(8). Epub 2021 Aug 23.

Dipartimento di Scienze Mediche Traslazionali, Università degli Studi di Napoli "Federico II", 80131 Napoli, Italy.

Introduction: Familial hypercholesterolemia (FH) is characterized by extremely high levels of circulating low-density lipoprotein cholesterol (LDL-C) and is caused by mutations of genes involved in LDL-C metabolism, including LDL receptor (LDLR), apolipoprotein B (APOB), or proprotein convertase subtilisin/Kexin type 9 (PCSK9). Accordingly, PCSK9 inhibitors (PCSK9i) are effective in LDL-C reduction. However, no data are available on the pleiotropic effect of PCSK9i. To this end, we performed an untargeted metabolomics approach to gather a global view on changes in metabolic pathways in patients receiving treatment with PCSK9i.

Methods: Twenty-five FH patients starting treatment with PCSK-9i were evaluated by an untargeted metabolomics approach at baseline (before PCSK9i treatment) and after 12 weeks of treatment.

Results: All the 25 FH subjects enrolled were on maximal tolerated lipid-lowering therapy prior to study entry. After a 12 week treatment with PCSK9i, we observed an expected significant reduction in LDL-cholesterol levels (from 201.0 ± 69.5 mg/dL to 103.0 ± 58.0 mg/dL, < 0.001). The LDL-C target was achieved in 36% of patients. After peak validation and correction, after 12 weeks of PCSK9i treatment as compared to baseline, we observed increments in creatine (-value = 0.041), indole (-value = 0.045), and indoleacrylic acid (-value= 0.045) concentrations. Conversely, significant decreases in choline (-value = 0.045) and phosphatidylcholine (-value < 0.01) together with a reduction in platelet activating factor (-value = 0.041) were observed.

Conclusions: Taking advantage of untargeted metabolomics, we first provided evidence of concomitant reductions in inflammation and platelet activation metabolites in FH patients receiving a 12 week treatment with PCSK9i.
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http://dx.doi.org/10.3390/biomedicines9081073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8391636PMC
August 2021

Aortic Valve Sclerosis in High-Risk Coronary Artery Disease Patients.

Front Cardiovasc Med 2021 27;8:711899. Epub 2021 Jul 27.

Centro Cardiologico Monzino Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Milan, Italy.

Current knowledge regarding the relationship between aortic valve sclerosis (AVSc), cardiovascular risk factors, and mortality in patients with known coronary artery disease (CAD) is still unclear. The present study aimed at investigating the prevalence of AVSc as well as its association with long-term all-cause mortality in high-risk CAD patients that has never been explored in large cohorts thus far. In this retrospective and observational cohort study we enrolled high-risk CAD patients, hospitalized at Centro Cardiologico Monzino (CCM), Milan, Italy, between January 2006 and December 2016. The morphology and function of the aortic valve were assessed from the recorded echocardiographic images to evaluate the presence of AVSc, defined as a non-uniform thickening of the aortic leaflets with no consequences on hemodynamics. Data on 5-year all-cause mortality was retrieved from a Regional database. Of the 5,489 patients initially screened, 4,938 (mean age 67 ± 11 years, 3,954 [80%] men) were enrolled in the study. In the overall population, AVSc was detected in 2,138 (43%) patients. Multivariable LASSO regression revealed that age, female gender, diabetes mellitus, previous MI, and left ventricular ejection fraction were independently associated with AVSc. All-cause mortality (adjusted hazard ratio: 1.29, 95%CI: 1.05-1.58) was significantly higher in AVSc than in non-AVSc patients. AVSc is frequently detected in high-risk CAD patients and is associated with long-term mortality. Our findings corroborate the hypothesis that AVSc is an underestimated marker of systemic cardiovascular risk. Thus, AVSc detection may be used to improve long-term risk stratification of high-risk CAD patients.
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http://dx.doi.org/10.3389/fcvm.2021.711899DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8354333PMC
July 2021

Coronary Artery Disease: Association Study of 5 Loci with Angiographic Indices of Disease Severity.

Dis Markers 2021 12;2021:5522539. Epub 2021 Jul 12.

Department of Cardiac Surgery, Centro Cardiologico, Monzino, Milan, Italy.

Background: Different common gene variants were related to coronary artery disease (CAD) in many studies. Yet, the relation of these loci to the severity of CAD is not completely elucidated.

Methods: We enrolled 520 subjects (315 CAD cases and 205 controls). CAD presence and extension were assessed by coronary angiography (CAG). Genotyping of five SNPs (namely, rs2230806 (1051G > A) in ABCA1 on chromosome 9, rs2075291 (553G > T) in ApoA5 on chromosome 11, rs320 in LPL on chromosome 8 intron (T → G at position 481), rs10757278 (c.22114477A > G), and rs2383206 (c.22115026 A > G) on chromosome 9p21 locus) was performed by allele-specific PCR. The degree and site of arterial lesions were used to classify patients, tested for association with CAD severity, and related to allele dosage.

Results: The polymorphisms rs2383206 and rs10757278 showed significant associations with 2- and 3-vessel coronary disease (p =0.003 and 0.006, respectively). The homozygous GG genotypes of rs10757278 was associated with higher frequency of left anterior descending (LAD), right coronary artery (RCA) and left circumflex (LCX) diseases (p =0.002, 0.016 and 0.002, respectively). The GG genotypes of rs2383206 were found in higher percentage in patients with left main (LM) trunk and left circumflex (LCX) diseases ( = 0.013 and 0.002, respectively).

Conclusion: SNPs rs10757278 and rs2383206 allele dosage could predict CAD severity in the Saudi Arab population.
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http://dx.doi.org/10.1155/2021/5522539DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8292076PMC
January 2022

Soluble Receptor for Advanced Glycation End-products regulates age-associated Cardiac Fibrosis.

Int J Biol Sci 2021 11;17(10):2399-2416. Epub 2021 Jun 11.

Unit of Experimental Cardio-Oncology and Cardiovascular Aging, Centro Cardiologico Monzino-IRCCS, Milan, Italy.

Myocardial aging increases the cardiovascular risk in the elderly. The Receptor for Advanced Glycation End-products (RAGE) is involved in age-related disorders. The soluble isoform (sRAGE) acts as a scavenger blocking the membrane-bound receptor activation. This study aims at investigating RAGE contribution to age-related cardiac remodeling. We analyzed the cardiac function of three different age groups of female and C57BL/6N (WT) mice: 2.5- (Young), 12- (Middle-age, MA) and 21-months (Old) old. While aging, mice displayed an increase in left ventricle (LV) dimensions compared to age-matched WT animals, with the main differences observed in the MA groups. mice showed higher fibrosis and a larger number of α-Smooth Muscle Actin (SMA)+ cells with age, along with increased expression of pro-fibrotic Transforming Growth Factor (TGF)-β1 pathway components. RAGE isoforms were undetectable in LV of WT mice, nevertheless, circulating sRAGE declined with aging and inversely associated with LV diastolic dimensions. Human cardiac fibroblasts stimulated with sRAGE exhibited a reduction in proliferation, pro-fibrotic proteins and TGF-beta Receptor 1 (TGFbR1) expression and Smad2-3 activation. Finally, sRAGE administration to MA WT animals reduced cardiac fibrosis. Hence, our work shows that RAGE associates with age-dependent myocardial changes and indicates sRAGE as an inhibitor of cardiac fibroblasts differentiation and age-dependent cardiac fibrosis.
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http://dx.doi.org/10.7150/ijbs.56379DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8315019PMC
March 2022

Evaluation of Oxford Nanopore MinION RNA-Seq Performance for Human Primary Cells.

Int J Mol Sci 2021 Jun 12;22(12). Epub 2021 Jun 12.

Centro Cardiologico Monzino IRCCS, 20131 Milan, Italy.

Transcript sequencing is a crucial tool for gaining a deep understanding of biological processes in diagnostic and clinical medicine. Given their potential to study novel complex eukaryotic transcriptomes, long-read sequencing technologies are able to overcome some limitations of short-read RNA-Seq approaches. Oxford Nanopore Technologies (ONT) offers the ability to generate long-read sequencing data in real time via portable protein nanopore USB devices. This work aimed to provide the user with the number of reads that should be sequenced, through the ONT MinION platform, to reach the desired accuracy level for a human cell RNA study. We sequenced three cDNA libraries prepared from poly-adenosine RNA of human primary cardiac fibroblasts. Since the runs were comparable, they were combined in a total dataset of 48 million reads. Synthetic datasets with different sizes were generated starting from the total and analyzed in terms of the number of identified genes and their expression levels. As expected, an improved sensitivity was obtained, increasing the sequencing depth, particularly for the non-coding genes. The reliability of expression levels was assayed by (i) comparison with PCR quantifications of selected genes and (ii) by the implementation of a user-friendly multiplexing method in a single run.
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http://dx.doi.org/10.3390/ijms22126317DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8231517PMC
June 2021

Lipidomics analysis of monocytes from patients with acute myocardial infarction reveals lactosylceramide as a new player in monocyte migration.

FASEB J 2021 05;35(5):e21494

Centro Cardiologico Monzino I.R.C.C.S, Milan, Italy.

Monocyte recruitment after vascular injury and their migration through the vessel wall represent crucial events in the initiation, progression, and destabilization of atherosclerotic plaque. Circulating monocytes are exposed to stimuli that alter their physiological state, and among them, lipids play a key role. Several studies investigated the mechanisms by which lipids affect monocyte functions promoting coronary atherosclerotic plaque initiation, but information on the relationship between lipid composition and function of monocyte is scant. We aimed at studying the migration of circulating monocytes isolated from patients with acute myocardial infarction (AMI) at hospital presentation and investigating its correlation with cellular lipid profile. The migration of monocytes was tested using both fetal bovine serum (FBS) and autologous serum as chemoattractant stimuli. Monocyte lipid profile was evaluated through an untargeted lipidomics approach, using a liquid chromatography/time-of-flight mass spectrometry platform. We observed that AMI patients' monocytes showed a significant increase in FBS and autologous serum-mediated migration compared to controls. Moreover, a different monocyte lipidomic profile between the two study groups was detected. In particular, AMI patients' monocytes showed an altered composition in ceramides, with an increase in lactosylceramide and in phospholipids (ie, phosphatidylethanolamine and lisophosphatidylethanolamine). Of note, a positive correlation between lactosylceramide levels and monocyte migration was observed. Furthermore, the lactosylceramide synthase inhibition significantly reduced FBS-induced monocyte migration. Our results highlight the influence of lactosylceramide on the monocyte migration capacity, pointing out a new possible mechanism of lipids in the onset of atherothrombosis and, hence, in AMI.
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http://dx.doi.org/10.1096/fj.202001872RRRDOI Listing
May 2021

The Association between HDL-C and Subclinical Atherosclerosis Depends on CETP Plasma Concentration: Insights from the IMPROVE Study.

Biomedicines 2021 Mar 11;9(3). Epub 2021 Mar 11.

Centro Cardiologico Monzino IRCCS, 20138 Milan, Italy.

The impact of cholesteryl ester transfer protein (CETP) on atherosclerosis is highly debated. This study aimed to investigate the associations between plasma CETP or CETP genotypes and carotid intima-media thickness (cIMT) and the influence of high-density lipoprotein cholesterol (HDL-C) on these associations. Plasma CETP and HDL-C concentrations were measured in 552 subjects free of any pharmacological treatment from the IMPROVE cohort, which includes 3711 European subjects at high cardiovascular risk. CETP single-nucleotide polymorphisms (SNPs) and cIMT measures (cIMT; cIMT of bifurcations, common and internal carotids; plaque-free common carotid [PF CC]-IMT) were available for the full cohort. In drug-free subjects, plasma CETP correlated with HDL-C levels (r = 0.19, < 0.0001), but not with cIMT variables. When stratified according to HDL-C quartiles, CETP positively correlated with cIMT and cIMT, but not with PF CC-IMT, in the top HDL-C quartile only. Positive associations between the CETP concentration and cIMT or cIMT were found in the top HDL-C quartile, whereas HDL-C levels were negatively correlated with cIMT and cIMT when the CETP concentration was below the median (HDL-C × CETP interaction, = 0.001 and = 0.003 for cIMT and cIMT, respectively). In the full cohort, three CETP SNPs (rs34760410, rs12920974, rs12708968) were positively associated with cIMT. rs12444708 exhibited a significant interaction with HDL-C levels in the prediction of cIMT. In conclusion, a significant interplay was found between plasma CETP and/or CETP genotype and HDL-C in the prediction of carotid plaque thickness, as indexed by cIMT. This suggests that the association of HDL-C with carotid atherosclerosis is CETP-dependent.
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http://dx.doi.org/10.3390/biomedicines9030286DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7999018PMC
March 2021

Digital PCR for high sensitivity viral detection in false-negative SARS-CoV-2 patients.

Sci Rep 2021 02 22;11(1):4310. Epub 2021 Feb 22.

Unità di Ingegneria Tissutale Cardiovascolare, Centro Cardiologico Monzino, IRCCS, Via Carlo Parea, 4, 20138, Milan, Italy.

Patients requiring diagnostic testing for coronavirus disease 2019 (COVID-19) are routinely assessed by reverse-transcription quantitative polymerase chain reaction (RT-qPCR) amplification of Sars-CoV-2 virus RNA extracted from oro/nasopharyngeal swabs. Despite the good specificity of the assays certified for SARS-CoV-2 molecular detection, and a theoretical sensitivity of few viral gene copies per reaction, a relatively high rate of false negatives continues to be reported. This is an important challenge in the management of patients on hospital admission and for correct monitoring of the infectivity after the acute phase. In the present report, we show that the use of digital PCR, a high sensitivity method to detect low amplicon numbers, allowed us to correctly detecting infection in swab material in a significant number of false negatives. We show that the implementation of digital PCR methods in the diagnostic assessment of COVID-19 could resolve, at least in part, this timely issue.
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http://dx.doi.org/10.1038/s41598-021-83723-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7900100PMC
February 2021

Classification of Psychoses Based on Immunological Features: A Machine Learning Study in a Large Cohort of First-Episode and Chronic Patients.

Schizophr Bull 2021 07;47(4):1141-1155

Department of Pathophysiology and Transplantation, University of Milan, Milan, Italy.

For several years, the role of immune system in the pathophysiology of psychosis has been well-recognized, showing differences from the onset to chronic phases. Our study aims to implement a biomarker-based classification model suitable for the clinical management of psychotic patients. A machine learning algorithm was used to classify a cohort of 362 subjects, including 160 first-episode psychosis patients (FEP), 70 patients affected by chronic psychiatric disorders (schizophrenia, bipolar disorder, and major depressive disorder) with psychosis (CRO) and 132 health controls (HC), based on mRNA transcript levels of 56 immune genes. Models distinguished between FEP, CRO, and HC and between the subgroup of drug-free FEP and HC with a mean accuracy of 80.8% and 90.4%, respectively. Interestingly, by using the feature importance method, we identified some immune gene transcripts that contribute most to the classification accuracy, possibly giving new insights on the immunopathogenesis of psychosis. Therefore, our results suggest that our classification model has a high translational potential, which may pave the way for a personalized management of psychosis.
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http://dx.doi.org/10.1093/schbul/sbaa190DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8266656PMC
July 2021

Differential Role of Circulating microRNAs to Track Progression and Pre-Symptomatic Stage of Chronic Heart Failure: A Pilot Study.

Biomedicines 2020 Dec 11;8(12). Epub 2020 Dec 11.

Immunology and Functional Genomics Unit, Centro Cardiologico Monzino-IRCCS, 20138 Milan, Italy.

(1)Background: Chronic heart failure (CHF) contributes to the overall burden of cardiovascular disease. Early identification of at-risk individuals may facilitate the targeting of precision therapies. Plasma microRNAs are promising circulating biomarkers for their implications with cardiac pathologies. In this pilot study, we investigate the possible exploitability of circulating micro-RNAs (miRNAs) to track chronic heart failure (CHF) occurrence, and progression from NYHA class I to IV. (2)Methods: We screened 367 microRNAs using TaqMan microRNA Arrays in plasma samples from healthy controls (HC) and CHF NYHA-class I-to-IV patients (5/group). Validation was performed by singleplex assays on 10 HC and 61 CHF subjects. Differences in the expression of validated microRNAs were evaluated through analysis of covariance (ANCOVA). Associations between N-terminal pro-BNP (NT-proBNP), left ventricular end-diastolic volume (LVEDV) or peak oxygen uptake (VO2 peak) and plasma microRNA were assessed by multivariable linear regression analysis. (3)Results: Twelve microRNAs showed higher expression in CHF patients vs. HC. Seven microRNAs were associated with NT-proBNP concentration; of these, miR-423-5p was also an independent predictor of LVEDV. Moreover, miR-499-5p was a predictor of the VO2 peak. Finally, a cluster of 5 miRNAs discriminated New York Heart Association (NYHA) class-I from HC subjects. (4)Conclusions: Our data suggest that circulating miRNAs have the potential to serve as pathophysiology-based markers of HF status and progression, and as indicators of pre-symptomatic individuals.
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http://dx.doi.org/10.3390/biomedicines8120597DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7764340PMC
December 2020

Circulating 16S RNA in Biofluids: Extracellular Vesicles as Mirrors of Human Microbiome?

Int J Mol Sci 2020 Nov 25;21(23). Epub 2020 Nov 25.

Unit of Immunology and Functional Genomics, Centro Cardiologico Monzino-IRCCS, 20138 Milan, Italy.

The human body is inhabited by around 10 microbes composing a multicomplex system, termed microbiota, which is strongly involved in the regulation and maintenance of homeostasis. Perturbations in microbiota composition can lead to dysbiosis, which has been associated with several human pathologies. The gold-standard method to explore microbial composition is next-generation sequencing, which involves the analysis of 16S rRNA, an indicator of the presence of specific microorganisms and the principal tool used in bacterial taxonomic classification. Indeed, the development of 16S RNA sequencing allows us to explore microbial composition in several environments and human body districts and fluids, since it has been detected in "germ-free" environments such as blood, plasma, and urine of diseased and healthy subjects. Recently, prokaryotes showed to generate extracellular vesicles, which are known to be responsible for shuttling different intracellular components such as proteins and nucleic acids (including 16S molecules) by protecting their cargo from degradation. These vesicles can be found in several human biofluids and can be exploited as tools for bacterial detection and identification. In this review, we examine the complex link between circulating 16S RNA molecules and bacteria-derived vesicles.
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http://dx.doi.org/10.3390/ijms21238959DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7728300PMC
November 2020

Plasma phospholipid dysregulation in patients with cystathionine-β synthase deficiency.

Nutr Metab Cardiovasc Dis 2020 11 23;30(12):2286-2295. Epub 2020 Jul 23.

Dipartimento di Scienze Mediche Traslazionali, Università degli Studi di Napoli "Federico II", 80131 Napoli, Italy.

Background & Aims: Patients with cystathionine β-synthase deficiency (CBSD) exhibit high circulating levels of homocysteine and enhanced lipid peroxidation. We have characterized the plasma lipidome in CBSD patients and related lipid abnormalities with reactions underlying enhanced homocysteine levels.

Methods And Results: Using an ultra-high-performance liquid chromatography-electrospray ionization-quadrupole-time of flight-mass spectrometry method, plasma lipids were determined with an untargeted lipidomics approach in 11 CBSD patients and 11 matched healthy subjects (CTRL). Compared to CTRL, CBSD patients had a higher medium and long-chain polyunsaturated fatty acids (PUFA) content in phosphatidylethanolamine (PE) and lysophosphatidylethanolamine (LPE) species (p < 0.02), and depletion of phosphatidylcholine (PC; p = 0.02) and of lysophosphatidylcholine (LPC; p = 0.003) species containing docosahexaenoic acid (DHA), suggesting impaired phosphatidylethanolamine-N-methyltransferase (PEMT) activity. PEMT converts PE into PC using methyl group by S-adenosylmethionine (SAM) thus converted in S-adenosylhomocysteine (SAH). Whole blood SAM and SAH concentrations by liquid chromatography tandem mass spectrometry were 1.4-fold (p = 0.015) and 5.3-fold (p = 0.003) higher in CBSD patients than in CTRL. A positive correlation between SAM/SAH and PC/PE ratios (r = 0.520; p = 0.019) was found.

Conclusions: A novel biochemical abnormality in CBSD patients consisting in depletion of PC and LPC species containing DHA and accumulation of PUFA in PE and LPE species is revealed by this lipidomic approach. Changes in plasma SAM and SAH concentrations are associated with such phospholipid dysregulation. Given the key role of DHA in thrombosis prevention, depletion of PC species containing DHA in CBSD patients provides a new direction to understand the poor cardiovascular outcome of patients with homocystinuria.
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http://dx.doi.org/10.1016/j.numecd.2020.07.019DOI Listing
November 2020

Sex-specific predictors of PCSK9 levels in a European population: The IMPROVE study.

Atherosclerosis 2020 09 30;309:39-46. Epub 2020 Jul 30.

Centro Cardiologico Monzino, IRCCS, Milan, Italy; Department of Medical Biotechnology and Translational Medicine, Università Degli Studi di Milano, Milan, Italy. Electronic address:

Background And Aims: Proprotein convertase subtilisin/kexin type 9 (PCSK9) is one of the key regulators of low-density lipoprotein cholesterol plasma levels. Circulating PCSK9, which differs between genders, represents a valid pharmacological target for preventing cardiovascular (CV) events. We aimed to investigate sex-related associations between PCSK9 plasma levels and biochemical and anthropomorphic factors, and familial and personal morbidities, in a large European cohort (n = 3673) of men (47.9%) and women (52.1%).

Methods: Individuals (aged 54-79 years) free of CV diseases were enrolled in seven centers of five European countries: Finland, France, Italy, the Netherlands, and Sweden. PCSK9 plasma levels were measured by ELISA.

Results: PCSK9 was higher in women than in men. Multiple linear regression analysis showed that latitude, sex, and treatments with statins and fibrates were the strongest predictors of PCSK9 in the whole group. These variables, together with triglycerides and high-density lipoprotein cholesterol, were also associated with PCSK9 in men or women. Mean corpuscular hemoglobin concentration and pack-years were PCSK9 independent predictors in women, whereas hypercholesterolemia and physical activity were independent predictors in men. The associations between PCSK9 and latitude, uric acid, diabetes, hypercholesterolemia and physical activity were significantly different in men and women (p <0.05 for all).

Conclusions: Besides confirming the association with lipids in the whole group, our study revealed previously unknown differences in PCSK9 predictors in men and women. These might be taken into account when defining individual risk for CV events and/or for refining PCSK9 lowering treatments.
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http://dx.doi.org/10.1016/j.atherosclerosis.2020.07.014DOI Listing
September 2020

Cardiac arrhythmia catheter ablation procedures guided by x-ray imaging: N-acetylcysteine protection against radiation-induced cellular damage (CARAPACE study): study design.

J Interv Card Electrophysiol 2021 Sep 24;61(3):577-582. Epub 2020 Aug 24.

Heart Rhythm Center, Centro Cardiologico Monzino IRCCS, Milan, Italy.

Purpose: Catheter ablation (CA) procedures are characterized by exposure to ionizing radiations (IR). IR can cause DNA damage and may lead to carcinogenesis if not efficiently repaired. The primary endpoint of this study is to investigate whether intravenous administration of N-acetylcysteine prior to CA procedure may prevent systemic oxidative stress and genomic DNA damage induced by exposure to IR.

Methods: The "Cardiac Arrhythmia catheter ablation procedures guided by x-Ray imaging: N-Acetylcysteine Protection Against radiation induced Cellular damagE" (CARAPACE) study is a prospective, randomized, single-blinded, parallel-arm monocenter study enrolling 550 consecutive patients undergoing CA at the Arrhythmology Unit of Centro Cardiologico Monzino (CCM). Inclusion criteria are age ≥ 18, indication for CA procedure guided by IR imaging, and written informed consent. IR levels will be measured via fluoroscopy time, effective dose, and dose area product. Glutathione and glutathione disulfide concentrations will be measured, and urinary levels of 8-iso-prostaglandin-F and 8-hydroxy-2-deoxyguanosine will be quantified. The enrolled patients will be randomized 1:1 to the N-acetylcysteine group or to the control group.

Results: We expect that pre-operative administration of N-acetylcysteine will prevent IR-induced systemic oxidative stress. The study will provide data on oxidative DNA damage assessed by urinary 8-hydroxy-2-deoxyguanosine levels and direct evidence of genomic DNA damage in blood cells by comet assay.

Conclusion: Catheter ablation procedures can lead to IR exposure and subsequent DNA damage. N-acetylcysteine administration prior to the procedure may prevent them and therefore lead to less possible complications.

Trial Registration: www.clinicaltrials.gov (NCT04154982).
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http://dx.doi.org/10.1007/s10840-020-00853-4DOI Listing
September 2021

Whole blood transcriptome profile at hospital admission discriminates between patients with ST-segment elevation and non-ST-segment elevation acute myocardial infarction.

Sci Rep 2020 05 26;10(1):8731. Epub 2020 May 26.

Unit of Immunology and Functional Genomics, Centro Cardiologico Monzino IRCCS, Milano, Italy.

Whether ST-segment (STEMI) and non-ST-segment elevation myocardial infarction (NSTEMI) should be regarded as distinct pathophysiological entities is a matter of debate. We tested the hypothesis that peripheral blood gene-expression profiles at presentation distinguish STEMI from NSTEMI. We performed a case-control study collecting whole-blood from 60 STEMI and 58 NSTEMI (defined according to the third universal definition of MI) consecutive patients on hospital admission. We used RNA-sequencing for the discovery phase, comparing 15 STEMI vs. 15 NSTEMI patients, matched for age, sex, and cardiovascular risk factors, and quantitative PCR in the remaining unmatched patients for validating top-significant genes. Gene-level differential expression analysis identified significant differences in the expression of 323 genes: 153 genes withstood correction for admission cardiac troponin I (cTnI), differentiating the two conditions independently of myocardial necrosis extent. Functional annotation analysis uncovered divergent modulation in leukocyte and platelet activation, cell migration, and mitochondrial respiratory processes. Linear regression analysis revealed gene expression patterns on admission predicting infarct size, as indexed by cTnI peak (R = 0.58-0.75). Our results unveil distinctive pathological traits for these two MI subtypes and provide insights into the early assessment of injury extent. This could translate into RNA-based disease-specific biomarkers for precision diagnosis and risk stratification.
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http://dx.doi.org/10.1038/s41598-020-65527-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7250845PMC
May 2020

Circulating MicroRNAs as Potential Predictors of Anthracycline-Induced Troponin Elevation in Breast Cancer Patients: Diverging Effects of Doxorubicin and Epirubicin.

J Clin Med 2020 May 11;9(5). Epub 2020 May 11.

Immunology and Functional Genomics Unit, Centro Cardiologico Monzino-IRCCS, 20138 Milan, Italy.

Anthracyclines are anti-neoplastic drugs presenting cardiotoxicity as a side effect. Cardiac troponins (cTn) and echocardiography are currently used to assess cardiac damage and dysfunction, but early biomarkers identifying patients in need of preventive treatments remain a partially met need. Circulating microRNAs (miRNAs) represent good candidates, so we investigated their possible roles as predictors of troponin elevation upon anthracycline treatment. Eighty-eight female breast cancer patients administered with doxorubicin (DOX) or epirubicin (EPI) were divided into four groups basing on drug type and cTn positive (cTn+) or negative (cTn-) levels: DOX cTn-, DOX cTn+, EPI cTn- and EPI cTn+. Blood was collected at baseline, during treatment, and at follow-up. We identified plasma miRNAs of interest by OpenArray screening and single assay validation. Our results showed miR-122-5p, miR-499a-5p and miR-885-5p dysregulation in DOX patients at T0, identifying a signature separating, with good accuracy, DOX cTn- from DOX cTn+. No miRNAs showed differential expression in EPI subjects. Conversely, an anthracycline-mediated modulation (regardless of cTn) was observed for miR-34a-5p, -122-5p and -885-5p. Our study indicates specific circulating miRNAs as possible prediction markers for cardiac troponin perturbation upon anthracycline treatment. Indeed, our findings hint at the possible future use of plasma miRNAs to predict the cardiac responsiveness of patients to different anticancer agents.
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http://dx.doi.org/10.3390/jcm9051418DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7290665PMC
May 2020

GARS: Genetic Algorithm for the identification of a Robust Subset of features in high-dimensional datasets.

BMC Bioinformatics 2020 Feb 11;21(1):54. Epub 2020 Feb 11.

Unit of Immunology and Functional Genomics, Centro Cardiologico Monzino IRCCS, Via Carlo Parea, 4, 20138, Milan, Italy.

Background: Feature selection is a crucial step in machine learning analysis. Currently, many feature selection approaches do not ensure satisfying results, in terms of accuracy and computational time, when the amount of data is huge, such as in 'Omics' datasets.

Results: Here, we propose an innovative implementation of a genetic algorithm, called GARS, for fast and accurate identification of informative features in multi-class and high-dimensional datasets. In all simulations, GARS outperformed two standard filter-based and two 'wrapper' and one embedded' selection methods, showing high classification accuracies in a reasonable computational time.

Conclusions: GARS proved to be a suitable tool for performing feature selection on high-dimensional data. Therefore, GARS could be adopted when standard feature selection approaches do not provide satisfactory results or when there is a huge amount of data to be analyzed.
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http://dx.doi.org/10.1186/s12859-020-3400-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7014945PMC
February 2020

CBS-miRSeq: A comprehensive tool for accurate and extensive analyses of microRNA-sequencing data.

Comput Biol Med 2019 07 1;110:234-243. Epub 2019 Jun 1.

Unit of Immunology and Functional Genomics, Centro Cardiologico Monzino IRCCS, Via Carlo Parea, 4, 20138, Milano, Italy. Electronic address:

Several online and local tools have been developed to analyze microRNA-sequencing (miRNA-Seq) data, but usually they are limited by many factors including: inaccurate processing, lack of optimal parameterization, outdated references plus annotations, restrictions in uploading large datasets, and shortage of biological inferences. In this work, we have developed a fully customized bioinformatics analysis pipeline (Color and Base-Space miRNA-Seq - CBS-miRSeq) for the seamless processing of short-reads miRNA-Seq data. The pipeline has been designed using Bash, Perl, and R scripts. CBS-miRSeq includes modules for read pre- and post-processing (quality assessment, filtering, adapter trimming and mapping) and different types of downstream analyses (identification of miRNA variants (isomiRs), novel miRNA prediction, miRNA:mRNA interaction target prediction, robust differential miRNA analysis, and target gene functional analysis). In this manuscript, we show that re-analysis of two published datasets using the CBS-miRSeq pipeline leads to better performance and efficiency in terms of their pipelines set and biomarker discovery between two biological conditions.
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http://dx.doi.org/10.1016/j.compbiomed.2019.05.019DOI Listing
July 2019

G-CSF for Extensive STEMI.

Circ Res 2019 07 29;125(3):295-306. Epub 2019 May 29.

Vascular Biology and Regenerative Medicine Unit (B.B., G. Pompilio), Centro Cardiologico Monzino IRCCS, Milano, Italy.

Rationale: In the exploratory Phase II STEM-AMI (Stem Cells Mobilization in Acute Myocardial Infarction) trial, we reported that early administration of G-CSF (granulocyte colony-stimulating factor), in patients with anterior ST-segment-elevation myocardial infarction and left ventricular (LV) dysfunction after successful percutaneous coronary intervention, had the potential to significantly attenuate LV adverse remodeling in the long-term.

Objective: The STEM-AMI OUTCOME CMR (Stem Cells Mobilization in Acute Myocardial Infarction Outcome Cardiac Magnetic Resonance) Substudy was adequately powered to evaluate, in a population showing LV ejection fraction ≤45% after percutaneous coronary intervention for extensive ST-segment-elevation myocardial infarction, the effects of early administration of G-CSF in terms of LV remodeling and function, infarct size assessed by late gadolinium enhancement, and myocardial strain.

Methods And Results: Within the Italian, multicenter, prospective, randomized, Phase III STEM-AMI OUTCOME trial, 161 ST-segment-elevation myocardial infarction patients were enrolled in the CMR Substudy and assigned to standard of care (SOC) plus G-CSF or SOC alone. In 119 patients (61 G-CSF and 58 SOC, respectively), CMR was available at baseline and 6-month follow-up. Paired imaging data were independently analyzed by 2 blinded experts in a core CMR lab. The 2 groups were similar for clinical characteristics, cardiovascular risk factors, and pharmacological treatment, except for a trend towards a larger infarct size and longer symptom-to-balloon time in G-CSF patients. ANCOVA showed that the improvement of LV ejection fraction from baseline to 6 months was 5.1% higher in G-CSF patients versus SOC (P=0.01); concurrently, there was a significant between-group difference of 6.7 mL/m in the change of indexed LV end-systolic volume in favor of G-CSF group (P=0.02). Indexed late gadolinium enhancement significantly decreased in G-CSF group only (P=0.04). Moreover, over time improvement of global longitudinal strain was 2.4% higher in G-CSF patients versus SOC (P=0.04). Global circumferential strain significantly improved in G-CSF group only (P=0.006).

Conclusions: Early administration of G-CSF exerted a beneficial effect on top of SOC in patients with LV dysfunction after extensive ST-segment-elevation myocardial infarction in terms of global systolic function, adverse remodeling, scar size, and myocardial strain.

Clinical Trial Registration: URL: https://www.clinicaltrials.gov. Unique identifier: NCT01969890.
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http://dx.doi.org/10.1161/CIRCRESAHA.118.314617DOI Listing
July 2019

Melanocortin-1 Receptor Positively Regulates Human Artery Endothelial Cell Migration.

Cell Physiol Biochem 2019 ;52(6):1339-1360

Unit of Immunology and Functional Genomics, Centro Cardiologico Monzino IRCCS, Milan, Italy,

Background/aims: Melanocortin receptors (MCRs) belong to a hormonal signalling pathway with multiple homeostatic and protective actions. Microvascular and umbilical vein endothelial cells (ECs) express components of the melanocortin system, including the type 1 receptor (MC1R), playing a role in modulating inflammation and vascular tone. Since ECs exhibit a remarkable heterogeneity, we investigated whether human artery ECs express any functional MCR and whether its activation affects cell migration.

Methods: We used reverse transcription real-time PCR to examine the expression of melanocortin system components in primary human artery ECs. We assessed MC1R protein expression and activity by western blot, immunohistochemistry, cAMP production, and intracellular Ca²⁺ mobilization assays. We performed gap closure and scratch tests to examine cell migration after stimulation with alpha-melanocyte-stimulating hormone (α-MSH), the receptor highest-affinity natural ligand. We assessed differential time-dependent transcriptional changes in migrating cells by microarray analysis.

Results: We showed that human aortic ECs (HAoECs) express a functionally active MC1R. Unlike microvascular ECs, arterial cells did not express the α-MSH precursor proopiomelanocortin, nor produced the hormone. MC1R engagement with a single pulse of α-MSH accelerated HAoEC migration both in the directional migration assay and in the scratch wound healing test. This was associated with an enhancement in Ca²⁺ signalling and inhibition of cAMP elevation. Time-course genome-wide expression analysis in HAoECs undergoing directional migration allowed identifying dynamic co-regulation of genes involved in extracellular matrix-receptor interaction, vesicle-mediated trafficking, and metal sensing - which have all well-established influences on EC motility -, without affecting the balance between pro- and anticoagulant genes.

Conclusion: Our work broadens the knowledge on peripherally expressed MC1R. These results indicate that the receptor is constitutively expressed by arterial ECs and provide evidence of a novel homeostatic function for MC1R, whose activation may participate in preventing/healing endothelial dysfunction or denudation in macrovascular arteries.
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http://dx.doi.org/10.33594/000000094DOI Listing
May 2019

A Specific Circulating MicroRNA Cluster Is Associated to Late Differential Cardiac Response to Doxorubicin-Induced Cardiotoxicity .

Dis Markers 2018 9;2018:8395651. Epub 2018 Dec 9.

Immunology and Functional Genomics Unit, Centro Cardiologico Monzino IRCCS, Milan, Italy.

Background: Cardiotoxicity is a detrimental side effect of the anticancer drug doxorubicin (DOX), characterized by progressive heart dysfunction. Circulating microRNAs (miRNAs) are recognized as potential biomarkers of cardiac disease; thus, we aimed to investigate their association with late cardiotoxicity in an animal model of disease.

Methods: Twenty C57BL/6 female mice were administered with 24 mg/kg cumulative dose of DOX or saline during 2 weeks, followed by a recovery period of one month (T42). Echocardiography was performed at baseline and at T42, and plasma samples were collected at T42. The selection of all miRNAs of interest was conducted by literature overview and by screening, followed by RT-qPCR validation The analysis of cardiac function at T42 evidenced five DOX-treated animals indistinguishable (NoTox) from controls (CTRLs), while four presented heart impairment (Tox). Our analyses identified eight dysfunction-associated plasma miRNAs. In particular, seven miRNAs were found downregulated in comparison to CTRLs, miR-1-3p, miR-122-5p, miR-127-3p, miR-133a-3p, miR-215-5p, miR-455-3-p, and miR-499a-5p. Conversely, miR-34a-5p showed increased levels in Tox plasma samples. Noteworthy, we determined a cluster composed of miR-1-3p, miR-34a-5p, miR-133a-3p, and miR-499a-5p that distinguished with high-accuracy Tox from NoTox mice.

Conclusion: This is the first study indicating that, similarly to what is observed in patients, DOX-administered animals present a differential cardiac response to treatment. Moreover, our results indicate the presence of specific plasma miRNAs whose expression reflect the presence of cardiac dysfunction in response to drug-induced injury.
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http://dx.doi.org/10.1155/2018/8395651DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6304816PMC
April 2019

Plasmatic and chamber-specific modulation of cardiac microRNAs in an acute model of DOX-induced cardiotoxicity.

Biomed Pharmacother 2019 Feb 16;110:1-8. Epub 2018 Nov 16.

Immunology and Functional Genomics Unit, Centro Cardiologico Monzino IRCCS, Milan, Italy. Electronic address:

Background: Doxorubicin (DOX) is a chemotherapeutic drug limited in its usefulness by an adverse side effect, cardiotoxicity. The mechanisms leading to this detrimental occurrence are not completely clear, and lately many authors focused their attention on the possible role of microRNAs (miRNAs), small regulators of cardiovascular functions, in this phenomenon. Notably, these molecules recently emerged also as potential circulating biomarkers of several cardiac diseases. Thus, the aim of this study was the simultaneous investigation of circulating and cardiac tissue miRNAs expression upon DOX treatment in vivo.

Methods: Twenty C57BL/6 female mice were administered with 24 mg/Kg cumulative dose of DOX or saline (CTRL) for 2 weeks. Echocardiography was performed at baseline and at the end of treatment (T1). Plasma and heart samples were collected at T1, separating atria from left (LV) and right (RV) ventricles, and miRNAs expression was tested by RT-qPCR-based arrays. All putatively DOX-regulated candidates were then validated by single assays in vivo and then evaluated also in murine immortalized cardiomyocytes (HL-1) treated with 1 μM DOX for 24 h. In the end, bioinformatics target prediction was performed for all DOX-miRNAs.

Results: Cardiotoxicity onset was diagnosed upon impairment of six cardiac functional parameters in DOX-treated mice at T1. Samples collection, followed by screening and validation steps, identified eleven miRNAs dysregulated by the drug in plasma, while seven resulted as altered in separate heart chambers. Interestingly, miR-34a-5p and miR-451a showed a dysregulation in both plasma and tissue samples of DOX-administered animals, whereas five additional miRNAs presented chamber specific modulation. Of note, in vitro experiments showed a very modest overlap with in vivo results. Bioinformatics prediction analysis performed on miR-34a-5p and miR-451a identified several putative targets presenting no significant association with cardiotoxicity. Anyhow, the same analyses, conducted by combining all miRNAs regulated by DOX in each heart chamber, evidenced a possible dysregulation of the adherens junctions gene network, known to be involved in the onset and progression of dilated cardiomyopathy, an established detrimental side effect of the drug.

Conclusions: This is the first work investigating miRNAs regulation by DOX both in plasma and heart districts of treated animals. Our results indicate a strong association of miR-34a-5p and miR-451a to DOX-induced cardiotoxicity. In addition, the observed altered expression of diverse miRNAs in separated cardiac chambers hints at a specific response to the drug, implying the existence of different players and pathways leading to dysfunction onset.
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http://dx.doi.org/10.1016/j.biopha.2018.11.042DOI Listing
February 2019
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