Circulating levels of AGEs and soluble RAGE isoforms are associated with all-cause mortality and development of cardiovascular complications in type 2 diabetes: a retrospective cohort study

none10noopenSabbatinelli, Jacopo; Castiglione, Stefania; Macrì, Federica; Giuliani, Angelica; Ramini, Deborah; Vinci, Maria Cristina; Tortato, Elena; Bonfigli, Anna Rita; Olivieri, Fabiola; Raucci, AngelaSabbatinelli, Jacopo; Castiglione, Stefania; Macrì, Federica; Giuliani, Angelica; Ramini, Deborah; Vinci, Maria Cristina; Tortato, Elena; Bonfigli, Anna Rita; Olivieri, Fabiola; Raucci, Angel

The Association between Single Nucleotide Polymorphisms, including miR-499a Genetic Variants, and Dyslipidemia in Subjects Treated with Pharmacological or Phytochemical Lipid-Lowering Agents

none12noDisorders of lipoprotein metabolism are among the major risk factors for cardiovascular disease (CVD) development. Single nucleotide polymorphisms (SNPs) have been associated with the individual variability in blood lipid profile and response to lipid-lowering treatments. Here, we genotyped 34 selected SNPs located in coding genes related to lipid metabolism, inflammation, coagulation, and a polymorphism in the MIR499 gene-a microRNA previously linked to CVD-to evaluate the association with lipid trait in subjects with moderate dyslipidemia not on lipid-lowering treatment (Treatment-naïve (TN) cohort, n = 125) and in patients treated with statins (STAT cohort, n = 302). We also explored the association between SNPs and the effect of a novel phytochemical lipid-lowering treatment in the TN cohort. We found that 6 SNPs (in the MIR499, TNFA, CETP, SOD2, and VEGFA genes) were associated with lipid traits in the TN cohort, while no association was found with the response to twelve-week phytochemical treatment. In the STAT cohort, nine SNPs (in the MIR499, CETP, CYP2C9, IL6, ABCC2, PON1, IL10, and VEGFA genes) were associated with lipid traits, three of which were in common with the TN cohort. Interestingly, in both cohorts, the presence of the rs3746444 MIR499 SNP was associated with a more favorable blood lipid profile. Our findings could add information to better understand the individual genetic variability in maintaining a low atherogenic lipid profile and the response to different lipid-lowering therapies.openGiuliani, Angelica; Montesanto, Alberto; Matacchione, Giulia; Graciotti, Laura; Ramini, Deborah; Protic, Olga; Galeazzi, Roberta; Antonicelli, Roberto; Tortato, Elena; Bonfigli, Anna Rita; Sabbatinelli, Jacopo; Olivieri, FabiolaGiuliani, Angelica; Montesanto, Alberto; Matacchione, Giulia; Graciotti, Laura; Ramini, Deborah; Protic, Olga; Galeazzi, Roberta; Antonicelli, Roberto; Tortato, Elena; Bonfigli, Anna Rita; Sabbatinelli, Jacopo; Olivieri, Fabiol

Prognostic value of soluble ST2, high-sensitivity cardiac troponin, and NT-proBNP in type 2 diabetes: a 15-year retrospective study

Background: Patients with type 2 diabetes (T2DM) present an increased risk of cardiovascular (CV) disease and excess CV-related mortality. Beyond the established role of brain natriuretic peptide (BNP) and cardiac troponins (cTn), other non-cardiac-specific biomarkers are emerging as predictors of CV outcomes in T2DM. Methods: Serum levels of soluble suppression of tumorigenesis 2 (sST2), high-sensitivity (hs)-cTnI, and N-terminal (NT)-proBNP were assessed in 568 patients with T2DM and 115 healthy controls (CTR). Their association with all-cause mortality and the development of diabetic complications was tested in T2DM patients over a median follow-up of 16.8 years using Cox models and logistic regressions. Results: sST2 followed an increasing trend from CTR to uncomplicated T2DM patients (T2DM-NC) to patients with at least one complication (T2DM-C), while hs-cTnI was significantly higher in T2DM-C compared to CTR but not to T2DM-NC. A graded association was found between sST2 (HR 2.76 [95% CI 1.20-6.33] for ≥ 32.0 ng/mL and 2.00 [1.02-3.94] for 16.5-32.0 ng/mL compared to < 16.5 ng/mL, C-statistic = 0.729), NT-proBNP (HR 2.04 [1.90-4.55] for ≥ 337 ng/L and 1.48 [1.05-2.10] for 89-337 ng/L compared to < 89 ng/L, C-statistic = 0.741), and 15-year mortality in T2DM, whereas increased mortality was observed in patients with hs-cTnI ≥ 7.8 ng/L (HR 1.63 [1.01-2.62]). A 'cardiac score' based on the combination of sST2, hs-cTnI, and NT-proBNP was significantly associated with all-cause mortality (HR 1.35 [1.19-1.53], C-statistic = 0.739) and development of CV events. Conclusions: sST2, hs-cTnI, and NT-proBNP are associated with 15-year mortality and onset of CV events in T2DM. The long-term prognostic value of sST2 and its ability to track variables related to insulin resistance and associated metabolic disorders support its implementation into routine clinical practice

Long-term exposure of human endothelial cells to metformin modulates miRNAs and isomiRs.

Increasing evidence suggest that the glucose-lowering drug metformin exerts a valuable anti-senescence role. The ability of metformin to affect the biogenesis of selected microRNAs (miRNAs) was recently suggested. MicroRNA isoforms (isomiRs) are distinct variations of miRNA sequences, harboring addition or deletion of one or more nucleotides at the 5\u27 and/or 3\u27 ends of the canonical miRNA sequence. We performed a comprehensive analysis of miRNA and isomiR profile in human endothelial cells undergoing replicative senescence in presence of metformin. Metformin treatment was associated with the differential expression of 27 miRNAs (including miR-100-5p, -125b-5p, -654-3p, -217 and -216a-3p/5p). IsomiR analysis revealed that almost 40% of the total miRNA pool was composed by non-canonical sequences. Metformin significantly affects the relative abundance of 133 isomiRs, including the non-canonical forms of the aforementioned miRNAs. Pathway enrichment analysis suggested that pathways associated with proliferation and nutrient sensing are modulated by metformin-regulated miRNAs and that some of the regulated isomiRs (e.g. the 5\u27 miR-217 isomiR) are endowed with alternative seed sequences and share less than half of the predicted targets with the canonical form. Our results show that metformin reshapes the senescence-associated miRNA/isomiR patterns of endothelial cells, thus expanding our insight into the cell senescence molecular machinery

Circulating miR-320b and miR-483-5p levels are associated with COVID-19 in-hospital mortality

none28noThe stratification of mortality risk in COVID-19 patients remains extremely challenging for physicians, especially in older patients. Innovative minimally invasive molecular biomarkers are needed to improve the prediction of mortality risk and better customize patient management. In this study, aimed at identifying circulating miRNAs associated with the risk of COVID-19 in-hospital mortality, we analyzed serum samples of 12 COVID-19 patients by small RNA-seq and validated the findings in an independent cohort of 116 COVID-19 patients by qRT-PCR. Thirty-four significantly deregulated miRNAs, 25 downregulated and 9 upregulated in deceased COVID-19 patients compared to survivors, were identified in the discovery cohort. Based on the highest fold-changes and on the highest expression levels, 5 of these 34 miRNAs were selected for the analysis in the validation cohort. MiR-320b and miR-483-5p were confirmed to be significantly hyper-expressed in deceased patients compared to survived ones. Kaplan-Meier and Cox regression models, adjusted for relevant confounders, confirmed that patients with the 20% highest miR-320b and miR-483-5p serum levels had three-fold increased risk to die during in-hospital stay for COVID-19. In conclusion, high levels of circulating miR-320b and miR-483-5p can be useful as minimally invasive biomarkers to stratify older COVID-19 patients with an increased risk of in-hospital mortality.restrictedGiuliani, Angelica; Matacchione, Giulia; Ramini, Deborah; Di Rosa, Mirko; Bonfigli, Anna Rita; Sabbatinelli, Jacopo; Monsurrò, Vladia; Recchioni, Rina; Marcheselli, Fiorella; Marchegiani, Francesca; Piacenza, Francesco; Cardelli, Maurizio; Galeazzi, Roberta; Pomponio, Giovanni; Ferrarini, Alessia; Gabrielli, Armando; Baroni, Silvia Svegliati; Moretti, Marco; Sarzani, Riccardo; Giordano, Piero; Cherubini, Antonio; Corsonello, Andrea; Antonicelli, Roberto; Procopio, Antonio Domenico; Ferracin, Manuela; Bonafè, Massimiliano; Lattanzio, Fabrizia; Olivieri, FabiolaGiuliani, Angelica; Matacchione, Giulia; Ramini, Deborah; Di Rosa, Mirko; Bonfigli, Anna Rita; Sabbatinelli, Jacopo; Monsurrò, Vladia; Recchioni, Rina; Marcheselli, Fiorella; Marchegiani, Francesca; Piacenza, Francesco; Cardelli, Maurizio; Galeazzi, Roberta; Pomponio, Giovanni; Ferrarini, Alessia; Gabrielli, Armando; Baroni, Silvia Svegliati; Moretti, Marco; Sarzani, Riccardo; Giordano, Piero; Cherubini, Antonio; Corsonello, Andrea; Antonicelli, Roberto; Procopio, Antonio Domenico; Ferracin, Manuela; Bonafè, Massimiliano; Lattanzio, Fabrizia; Olivieri, Fabiol

Sensitivity and specificity of in vivo COVID-19 screening by detection dogs: Results of the C19-Screendog multicenter study

Trained dogs can recognize the volatile organic compounds contained in biological samples of patients with COVID-19 infection. We assessed the sensitivity and specificity of in vivo SARS-CoV- 2 screening by trained dogs. We recruited five dog-handler dyads. In the operant conditioning phase, the dogs were taught to distinguish between positive and negative sweat samples collected from volunteers’ underarms in polymeric tubes. The conditioning was validated by tests involving 16 positive and 48 negative samples held or worn in such a way that the samples were invisible to the dog and handler. In the screening phase the dogs were led by their handlers to a drive-through facility for in vivo screening of volunteers who had just received a nasopharyngeal swab from nursing staff. Each volunteer who had already swabbed was subsequently tested by two dogs, whose responses were recorded as positive, negative, or inconclusive. The dogs’ behavior was constantly monitored for attentiveness and wellbeing. All the dogs passed the conditioning phase, their responses showing a sensitivity of 83-100% and a specificity of 94-100%. The in vivo screening phase involved 1251 subjects, of whom 205 had a COVID-19 positive swab and two dogs per each subject to be screened. Screeningsensitivity and specificity were respectively 91.6-97.6% and 96.3-100% when only one dog was involved, whereas combined screening by two dogs provided a higher sensitivity. Dog wellbeing was also analysed: monitoring of stress and fatigue suggested that the screening activity did not adversely impact the dogs’ wellbeing. This work, by screening a large number of subjects, strengthen recent findings that trained dogs can discriminate between COVID-19 infected and healthy human subjects and introduce two novel research aspects: i) assessement of signs of fatigue and stress in dogs during training and testing, and ii) combining screening by two dogs to improve detection sensitivity and specificity. Using some precautions to reduce the risk of infection and spillover, in vivo COVID-19 screening by a dog-handler dyad can be suitable to quickly screen large numbers of people: it is rapid, non- invasiveand economical, since it does not involve actual sampling, lab resources or waste management, and is suitable to screen large numbers of people

Replicative Senescence-Associated LINE1 Methylation and LINE1-Alu Expression Levels in Human Endothelial Cells

One of the main challenges of current research on aging is to identify the complex epigenetic mechanisms involved in the acquisition of the cellular senescent phenotype. Despite some evidence suggested that epigenetic changes of DNA repetitive elements, including transposable elements (TE) sequences, are associated with replicative senescence of fibroblasts, data on different types of cells are scarce. We previously analysed genome-wide DNA methylation of young and replicative senescent human endothelial cells (HUVECs), highlighting increased levels of demethylated sequences in senescent cells. Here, we aligned the most significantly demethylated single CpG sites to the reference genome and annotated their localization inside TE sequences and found a significant hypomethylation of sequences belonging to the Long-Interspersed Element-1 (LINE-1 or L1) subfamilies L1M, L1P, and L1HS. To verify the hypothesis that L1 demethylation could be associated with increased transcription/activation of L1s and/or Alu elements (non-autonomous retroelements that usually depend on L1 sequences for reverse transcription and retrotransposition), we quantified the RNA expression levels of both L1 (generic L1 elements or site-specific L1PA2 on chromosome 14) and Alu elements in young and senescent HUVECs and human dermal fibroblasts (NHDFs). The RNA expression of Alu and L1 sequences was significantly increased in both senescent HUVECs and NHDFs, whereas the RNA transcript of L1PA2 on chromosome 14 was not significantly modulated in senescent cells. Moreover, we found an increased amount of TE DNA copies in the cytoplasm of senescent HUVECs and NHDFs. Our results support the hypothesis that TE, which are significantly increased in senescent cells, could be retrotranscribed to DNA sequences

The telomere world and aging: Analytical challenges and future perspectives

Telomeres, the terminal nucleoprotein structures of eukaryotic chromosomes, play pleiotropic functions in cellular and organismal aging. Telomere length (TL) varies throughout life due to the influence of genetic factors and to a complex balancing between “shortening” and “elongation” signals. Telomerase, the only enzyme that can elongate a telomeric DNA chain, and telomeric repeat-containing RNA (TERRA), a long non-coding RNA involved in looping maintenance, play key roles in TL during life. Despite recent advances in the knowledge of TL, TERRA and telomerase activity (TA) biology and their measurement techniques, the experimental and theoretical issues involved raise a number of problems that should carefully be considered by researchers approaching the “telomere world”. The increasing use of such parameters – hailed as promising clinically relevant biomarkers – has failed to be paralleled by the development of automated and standardized measurement technology. Consequently, associating given TL values to specific pathological conditions involves on the one hand technological issues and on the other clinical-biological issues related to the planning of clinically relevant association studies. Addressing these issues would help avoid major biases in association studies involving TL and a number of outcomes, especially those focusing on psychological and bio-behavioral variables. The main challenge in telomere research is the development of accurate and reliable measurement methods to achieve simple and sensitive TL, TERRA, and TA detection. The discovery of the localization of telomeres and TERRA in cellular and extracellular compartments had added an additional layer of complexity to the measurement of these age-related biomarkers. Since combined analysis of TL, TERRA and TA may well provide more exhaustive clinical information than a single parameter, we feel it is important for researchers in the various fields to become familiar with their most common measurement techniques and to be aware of the respective merits and drawbacks of these approache

Zingiber officinale Roscoe Rhizome Extract Exerts Senomorphic and Anti-Inflammatory Activities on Human Endothelial Cells

: Aging is related to a low-grade and sterile inflammation called inflammaging, recognized as the main risk factor for age-related disease (ARD) development. Inflammaging is fostered by the repeated activation of immune cells, as well as by the accumulation of senescent cells. Recently, a number of natural compounds have gained attention to be tested as anti-aging therapies, based on their anti-inflammatory activity and/or ability to reduce the pro-inflammatory secretome of senescent cells (senomorphyc activity). Here, we investigated the anti-inflammatory and senomorphic properties of an Asian-native Zingiber officinale Roscoe extract (ZOE), commonly consumed as a food spice and herbal medicine. We employed two models of primary endothelial cells (HUVECs), such as the replicative-senescence and LPS-induced response, to investigate the anti-inflammatory/senomorphic effect of ZOE, and one cellular model of neuroinflammation, i.e., immortalized murine microglial cells (BV2). First, we found that the ZOE treatment induced the inhibition of NF-kB activation in BV2 cells. Among the constituents of ZOE, we showed that the terpenoid-enriched fraction (ZTE) was the component able to counteract the phosphorylation of NF-kB(p65), while 6-gingerol (GIN) and 6-shogaol (SHO) did not produce any significant effect. Further, we observed that the treatment with 10 µg/mL of ZOE exerted anti-inflammatory activity on LPS-stimulated young (y)HUVEC and senomorphyc activity on replicative senescent (s)HUVEC, significantly reducing the expression levels of IL-1β, TNF -α, IL-8, MCP-1, and ICAM-1. Moreover, the ZTE treatment was able to significantly reduce the IL-8 levels secreted in the medium of both LPS-stimulated yHUVEC and sHUVEC. Overall, our data suggest a potential protective role of ZOE on neuroinflammation and endothelial inflammation/activation, thus suggesting its potential relevance in delaying/postponing ARD development and progression, characterized by endothelial dysfunction