Association of Inflammatory Mediators with Mitochondrial DNA Variants in Geriatric COVID-19 Patients.

COVID-19 remains a serious concern for elderly individuals with underlying comorbidities. SARS-CoV-2 can target and damage mitochondria, potentially leading to mutations in mitochondrial DNA (mtDNA). This study aimed to evaluate single nucleotide substitutions in mtDNA and analyze their correlation with inflammatory biomarkers in elderly COVID-19 patients. A total of 30 COVID-19 patients and 33 older adult controls without COVID-19 (aged over 65 years) were enrolled. mtDNA was extracted from buffy coat samples and sequenced using a chip-based resequencing system (MitoChip v2.0) which detects both homoplasmic and heteroplasmic mtDNA variants (40-60% heteroplasmy), and allows the assessment of low-level heteroplasmy (<10% heteroplasmy). Serum concentrations of IL-6, IFN-α, TNF-α and IL-10 were determined in patients by a high-sensitivity immunoassay. We found a higher burden of total heteroplasmic variants in COVID-19 patients compared to controls with a selective increment in ND1 and COIII genes. Low-level heteroplasmy was significantly elevated in COVID-19 patients, especially in genes of the respiratory complex I. Both heteroplasmic variant burden and low-level heteroplasmy were associated with increased levels of IL-6, TNF-α, and IFN-α. These findings suggest that SARS-CoV-2 may induce mtDNA mutations that are related to the degree of inflammation.

Low circulating levels of miR-17 and miR-126-3p are associated with increased mortality risk in geriatric hospitalized patients affected by cardiovascular multimorbidity.

MultiMorbidity (MM), defined as the co-occurrence of two or more chronic conditions, is associated with poorer health outcomes, such as recurrent hospital readmission and mortality. As a group of conditions, cardiovascular disease (CVD) exemplifies several challenges of MM, and the identification of prognostic minimally invasive biomarkers to stratify mortality risk in patients affected by cardiovascular MM is a huge challenge. Circulating miRNAs associated to inflammaging and endothelial dysfunction, such as miR-17, miR-21-5p, and miR-126-3p, are expected to have prognostic relevance. We analyzed a composite profile of circulating biomarkers, including miR-17, miR-21-5p, and miR-126-3p, and routine laboratory biomarkers in a sample of 246 hospitalized geriatric patients selected for cardiovascular MM from the Report-AGE INRCA database and BioGER INRCA biobank, to evaluate the association with all-cause mortality during 31 days and 12 and 24 months follow-up. Circulating levels of miR-17, miR-126-3p, and some blood parameters, including neutrophil to lymphocyte ratio (NLR) and eGFR, were significantly associated with mortality in these patients. Overall, our results suggest that in a cohort of geriatric hospitalized patients affected by cardiovascular MM, lower circulating miR-17 and miR-126-3p levels could contribute to identify patients at higher risk of short- and medium-term mortality.

Effect of Cytomegalovirus Reactivation on Inflammatory Status and Mortality of Older COVID-19 Patients.

Herpesviridae reactivation such as cytomegalovirus (CMV) has been described in severe COVID-19 (COronaVIrusDisease-2019). This study aimed to understand if CMV reactivation in older COVID-19 patients is associated with increased inflammation and in-hospital mortality. In an observational single-center cohort study, 156 geriatric COVID-19 patients were screened for CMV reactivation by RT-PCR. Participants underwent a comprehensive clinical investigation that included medical history, functional evaluation, laboratory tests and cytokine assays (TNF-α, IFN-α, IL-6, IL-10) at hospital admission. In 19 (12.2%) of 156 COVID-19 patients, CMV reactivation was detected. Multivariate Cox regression models showed that in-hospital mortality significantly increased among CMV positive patients younger than 87 years (HR: 9.94, 95% CI: 1.66-59.50). Other factors associated with in-hospital mortality were C-reactive protein (HR: 1.17, 95% CI: 1.05-1.30), neutrophil count (HR: 1.20, 95% CI: 1.01-1.42) and clinical frailty scale (HR:1.54, 95% CI: 1.04-2.28). In patients older than 87 years, neutrophil count (HR: 1.13, 95% CI: 1.05-1.21) and age (HR: 1.15, 95% CI: 1.01-1.31) were independently associated with in-hospital mortality. CMV reactivation was also correlated with increased IFN-α and TNF-α serum levels, but not with IL-6 and IL-10 serum changes. In conclusion, CMV reactivation was an independent risk factor for in-hospital mortality in COVID-19 patients younger than 87 years old, but not in nonagenarians.

Sensibility and Specificity of the VitaPCR™ SARS-CoV-2 Assay for the Rapid Diagnosis of COVID-19 in Older Adults in the Emergency Department.

(1) Background: During the COVID-19 pandemic, rapid and reliable diagnostic tools are needed for detecting SARS-CoV-2 infection in urgent cases at admission to the hospital. We aimed to assess the performances of the rapid molecular VitaPCR™ test (Menarini Diagnostics) in a sample of older adults admitted to the Emergency Department of two Italian hospitals (2) Methods: The comparison between the rapid VitaPCR™ and the RT-PCR was performed in 1695 samples. Two naso-pharyngeal swab samplings from each individual were obtained and processed using the VitaPCR™ and the RT-PCR for the detection of SARS-CoV-2 (3) Results: VitaPCR™ exhibited good precision (<3% CV) and an almost perfect overall agreement (Cohen's K = 0.90) with the RT-PCR. The limit of detection of the VitaPCR™ was 4.1 copies/µL. Compared to the RT-PCR, the sensitivity, the specificity, and the positive and negative predictive values of VitaPCR™ were 83.4%, 99.9%, 99.2% and 98.3%, respectively (4) Conclusions: The VitaPCR™ showed similar sensitivity and specificity to other molecular-based rapid tests. This study suggests that the VitaPCR™ can allow the rapid management of patients within the Emergency Department. Nevertheless, it is advisable to obtain a negative result by a RT-PCR assay before admitting a patient to a regular ward.

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

The 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.

Prognostic relevance of normocytic anemia in elderly patients affected by cardiovascular disease.

BACKGROUND: Anemia associated with cardiovascular diseases (CVD) is a common condition in older persons. Prevalence and prognostic role of anemia were extensively studied in patients with myocardial infarction (MI) or congestive heart failure (CHF) whereas limited data were available on patients with atrial fibrillation (AF). This study was conducted to assess the clinical prevalence and prognostic relevance of anemia in elderly patients affected by AF and other CVDs. METHODS: A total of 866 elderly patients (430 men and 436 women, age: 65-98 years, mean age: 85 ± 10 years) were enrolled. Among these patients, 267 patients had acute non-ST-segment elevation MI (NSTEMI), 176 patients had acute CHF, 194 patients had acute AF and 229 patients were aged-matched healthy persons (CTR). All parameters were measured at the hospital admission and cardiovascular mortality was assessed during twenty-four months of follow-up. RESULTS: The prevalence of anemia was higher in NSTEMI, CHF and AF patients compared to CTR subjects (50% vs. 15%, P < 0.05), with normocytic anemia being the most prevalent type (90%). Adjusted mortality risk was higher in anemic patient versus non-anemic patient in all the groups of patients [NSTEMI: hazard ratio (HR) = 1.81, 95% CI: 1.06-2.13; CHF: HR = 2.49, 95% CI: 1.31-4.75; AF: HR = 1.98, 95% CI: 1.01-3.88]. Decreased hemoglobin levels ( P = 0.001) and high reticulocyte index (P = 0.023) were associated with higher mortality in CVD patients. CONCLUSIONS: The significant associations between CVD and anemia and the prognostic relevance of anemia for elderly patients with CVD were confirmed in this study. The presence of anemia in AF patients is associated with a two-fold increased mortality risk compared with non-anemic AF patients. Low hemoglobin and high reticulocyte count independently predict mortality in elderly patients with CVD.

Anti-SASP and anti-inflammatory activity of resveratrol, curcumin and ß-caryophyllene association on human endothelial and monocytic cells.

A challenging and promising new branch of aging-related research fields is the identification of natural compounds able to modulate the senescence-associated secretory phenotype (SASP), which characterizes senescent cells and can contribute to fuel the inflammaging. We investigated both the anti-SASP and anti-inflammatory activities of a nutritional supplement, namely Fenoxidol™, composed of turmeric extract bioCurcumin (bCUR), Polydatin (the natural glycosylated precursor of Resveratrol-RSV), and liposomal ß-caryophyllene (BCP), in two human cellular models, such as the primary endothelial cell line, HUVECs and the monocytic cell line, THP-1. Replicative and Doxorubicin-induced senescent HUVECs, both chosen as cellular models of SASP, and lipopolysaccharides (LPS)-stimulated THP-1, selected as a model of the inflammatory response, were treated with the three single natural compounds or with a combination of them (MIX). In both senescent HUVEC models, MIX treatment significantly reduced IL-1ß and IL-6 expression levels and p16ink4a protein, and also increased SIRT1 protein level, as well as downregulated miR-146a and miR-21 expression, two of the so-called inflamma-miRNAs, more effectively than the single compounds. In THP-1 cells stimulated with LPS, the MIX showed a significant effect in decreasing IL-1ß, IL-6, TNF-α, and miR-146a expression levels and Caspase-1 activation, in association with an up-regulation of SIRT1 protein, compared to the single compounds. Overall, our results suggest that the three analysed compounds can have a combined effect in restraining SASP in senescent HUVECs as well as the inflammatory response in LPS-stimulated THP-1 cells.

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' and/or 3' 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' 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.

Small extracellular vesicles deliver miR-21 and miR-217 as pro-senescence effectors to endothelial cells.

The role of epigenetics in endothelial cell senescence is a cutting-edge topic in ageing research. However, little is known of the relative contribution to pro-senescence signal propagation provided by microRNAs shuttled by extracellular vesicles (EVs) released from senescent cells. Analysis of microRNA and DNA methylation profiles in non-senescent (control) and senescent (SEN) human umbilical vein endothelial cells (HUVECs), and microRNA profiling of their cognate small EVs (sEVs) and large EVs demonstrated that SEN cells released a significantly greater sEV number than control cells. sEVs were enriched in miR-21-5p and miR-217, which target DNMT1 and SIRT1. Treatment of control cells with SEN sEVs induced a miR-21/miR-217-related impairment of DNMT1-SIRT1 expression, the reduction of proliferation markers, the acquisition of a senescent phenotype and a partial demethylation of the locus encoding for miR-21. MicroRNA profiling of sEVs from plasma of healthy subjects aged 40-100 years showed an inverse U-shaped age-related trend for miR-21-5p, consistent with senescence-associated biomarker profiles. Our findings suggest that miR-21-5p/miR-217 carried by SEN sEVs spread pro-senescence signals, affecting DNA methylation and cell replication.

Three Months Monitored Metabolic Fitness Modulates Cardiovascular Risk Factors in Diabetic Patients.

Cardiovascular diseases represent the leading cause of death and moderate physical exercise is associated with a reduction in cardiovascular risk. The aim of the study was to evaluate the correlation between the amount of exercise recorded daily by a wearable gravitometer for 3 months and selected biochemical and clinical parameters. Nineteen sedentary type 2 diabetics were recruited and distributed into three homogenous groups, low, medium, and high exercise, according to the level of physical exercise monitored and expressed as MOVEs. Data showed an inverse correlation between MOVEs and oxidative stress indexes and a significant improvement in paraoxonase-1 activities and endothelial functionality. Decrease of visceral/total adipose tissue ratio, systolic blood pressure and a down-regulation of the inflammatory microRNA-146a in high exercise group were observed. Finally, a decrease of glycosylated hemoglobin and an up-regulation of the angiogenic microRNA-130a in medium exercise one was obtained. In this study, precise daily monitoring permitted to underline the importance of the amount of physical activity to counteract some cardiovascular risk factors persisting in diabetes. Finally, it identifies new microRNA biomarkers for future investigation on the same topic.

Diagnostic performance of new and classic CSF biomarkers in age-related dementias.

The identification of diagnostic-prognostic biomarkers of dementia has become a global priority due to the prevalence of neurodegenerative diseases in aging populations. The objective of this study was to assess the diagnostic performance of cerebrospinal fluid (CSF) biomarkers across patients affected by either Alzheimer's disease (AD), tauopathies other than AD (TP), or vascular dementia (VD), and cognitively normal subjects (CNS). One hundred fifty-three patients were recruited and tested for classical AD CSF biomarkers- Amyloid-ß42 and tau proteins - and novel candidate biomarkers - neurofilament (NF-) light and microRNA (miR) -21, -125b, -146a, and -222.All dementia patients had significantly higher concentrations of NF-light compared to CNS, with the TP group displaying the highest NF-light values. A significant inverse correlation was also observed between NF-light and cognitive impairment. Of the four miRNAs analyzed, miR-222 levels were significantly increased in VD patients compared to both CNS and AD. In addition, while NF-light showed a better diagnostic performance than miR-222 and classical AD biomarkers in differentiating TP and VD from CNS, classical AD biomarkers revealed higher performance in discriminating AD from non-AD disorders.Overall, our results suggest that CSF NF-light and miR-222 are promising biomarkers that may help to diagnose non-AD disorders.

Short-term sustained hyperglycaemia fosters an archetypal senescence-associated secretory phenotype in endothelial cells and macrophages.

Diabetic status is characterized by chronic low-grade inflammation and an increased burden of senescent cells. Recently, the senescence-associated secretory phenotype (SASP) has been suggested as a possible source of inflammatory factors in obesity-induced type 2 diabetes. However, while senescence is a known consequence of hyperglycaemia, evidences of SASP as a result of the glycaemic insult are missing. In addition, few data are available regarding which cell types are the main SASP-spreading cells in vivo. Adopting a four-pronged approach we demonstrated that: i) an archetypal SASP response that was at least partly attributable to endothelial cells and macrophages is induced in mouse kidney after in vivo exposure to sustained hyperglycaemia; ii) reproducing a similar condition in vitro in endothelial cells and macrophages, hyperglycaemic stimulus largely phenocopies the SASP acquired during replicative senescence; iii) in endothelial cells, hyperglycaemia-induced senescence and SASP could be prevented by SOD-1 overexpression; and iiii) ex vivo circulating angiogenic cells derived from peripheral blood mononuclear cells from diabetic patients displayed features consistent with the SASP. Overall, the present findings document a direct link between hyperglycaemia and the SASP in endothelial cells and macrophages, making the SASP a highly likely contributor to the fuelling of low-grade inflammation in diabetes.

Age-related modulation of plasmatic beta-Galactosidase activity in healthy subjects and in patients affected by T2DM.

ß-Galactosidase (ß-Gal) activity has been the most extensively utilized biomarker for the detection of cellular senescence. It can be measured also in plasma, and few recent evidence showed an altered plasmatic ß-Gal activity in patients affected by some age-related diseases (ARDs). Since T2DM is one of the most common ARDs, we aimed to investigate if plasmatic ß-Gal activity is modulated in T2DM patients and if "age" could affect such modulation. To gain mechanistic insights we paralleled this investigation with the evaluation of ß-Gal activity in young and senescent endothelial cells (HUVECs) cultured in normo- and hyper-glycaemic environment. A significant age-related increase of plasmatic ß-Gal activity was observed in healthy subjects (n. 230; 55-87 years), whereas the enzymatic activity was significantly reduced in T2DM patients (n. 230; 55-96 years) compared to healthy subjects. ß-Gal activity detectable both in cells and in the culture medium was significantly increased in senescent cells compared to the younger ones, both under normo- and hyper-glycaemic condition. However, the hyper-glycaemic condition was not associated with an increased ß-Gal activity in milieu compared to normo-glycaemic condition. Overall our data reinforce the notion that plasmatic ß-Gal activity could be a systemic biomarker of aging, whereas T2DM patients are characterized by a different age-releated trend.

Epigenetic effects of physical activity in elderly patients with cardiovascular disease.

Cardiovascular disease (CVD) is an important public health problem affecting especially the elderly. Over the past 20years, an increasing number of studies have examined its underlying pathophysiological mechanisms and new therapies are continually being discovered. However, despite considerable progress in CVD management, mortality and morbidity remain a major healthcare concern, and frequent hospital admissions compromise the daily life and social activities of these patients. Physical activity has emerged as an important non-pharmacological adjunctive therapy for CVD in older patients, especially for heart failure patients, exerting its beneficial effects on mortality, morbidity, and functional capacity. The mechanisms underlying the cardiovascular benefits of exercise are not wholly clear. Mounting evidence suggest that epigenetic modifications, such as DNA methylation, histone post-translational modifications (hPTMs) and non-coding RNA, especially microRNAs (miRNAs), may be induced by physical activity. Recently, a number of miRNAs have been identified as key players in gene expression modulation by exercise. MiRNAs are synthesized by living cells and actively released into the bloodstream through different shuttles. The epigenetic information, thus carried and delivered, is involved in the interplay between environmental factors, including physical activity, and individual genetic make-up. We review and discuss the effects of exercise on age-related CVDs, focusing on circulating miRNA (c-miRNAs) modulation. Epigenetic mechanisms may have clinical relevance in CVD prevention and management; since they can be modified, insights into the implications of lifestyle-related epigenetic changes in CVD etiology may help develop therapeutic protocols of exercise training that can be suitable and effective for elderly patients.

Systemic Age-Associated DNA Hypermethylation of ELOVL2 Gene: In Vivo and In Vitro Evidences of a Cell Replication Process.

Epigenetic remodeling is one of the major features of the aging process. We recently demonstrated that DNA methylation of ELOVL2 and FHL2 CpG islands is highly correlated with age in whole blood. Here we investigated several aspects of age-associated hypermethylation of ELOVL2 and FHL2. We showed that ELOVL2 methylation is significantly different in primary dermal fibroblast cultures from donors of different ages. Using epigenomic data from public resources, we demonstrated that most of the tissues show ELOVL2 and FHL2 hypermethylation with age. Interestingly, ELOVL2 hypermethylation was not found in tissues with very low replication rate. We demonstrated that ELOVL2 hypermethylation is associated with in vitro cell replication rather than with senescence. We confirmed intra-individual hypermethylation of ELOVL2 and FHL2 in longitudinally assessed participants from the Doetinchem Cohort Study. Finally we showed that, although the methylation of the two loci is not associated with longevity/mortality in the Leiden Longevity Study, ELOVL2 methylation is associated with cytomegalovirus status in nonagenarians, which could be informative of a higher number of replication events in a fraction of whole-blood cells. Collectively, these results indicate that ELOVL2 methylation is a marker of cell divisions occurring during human aging.

Physical activity and progenitor cell-mediated endothelial repair in chronic heart failure: Is there a role for epigenetics?

Chronic heart failure (CHF) is the most common cardiac disease among the elderly and a leading cause of mortality in elderly patients. Endothelial dysfunction is held to have a major role in the development and progression of CHF, which results in progressively impaired functional capacity. Endothelial progenitor cells (EPCs) and circulating angiogenic cells (CACs) are the main players involved in the endogenous repair mechanisms that can counteract endothelial dysfunction. A mounting body of data indicates that exercise enhances endothelial renewal through mobilization of bone marrow-derived EPCs and CACs, making it an effective therapeutic tool for CHF. Interestingly, emerging evidence has been showing that exercise training can also promote epigenetic modifications, e.g. DNA methylation, histone modifications, and differential expression of specific non-coding RNAs like microRNA (miRNAs). Since deregulation of the miRNAs involved in endothelial function modulation has widely been documented in circulating cells and plasma of CHF patients, deregulation of epigenetic features could play a key role in disease progression. Here, we review current knowledge of the contribution of EPCs and CACs to endothelial repair mechanisms in CHF patients, focusing on the effects induced by exercise training and hypothesizing that some of these effects can be mediated by epigenetic mechanisms.

Anti-TNF-α treatment modulates SASP and SASP-related microRNAs in endothelial cells and in circulating angiogenic cells.

Endothelial cell senescence is characterized by acquisition of senescence-associated secretory phenotype (SASP), able to promote inflammaging and cancer progression. Emerging evidence suggest that preventing SASP development could help to slow the rate of aging and the progression of age-related diseases, including cancer. Aim of this study was to evaluate whether and how adalimumab, a monoclonal antibody directed against tumor necrosis factor-α (TNF-α), a major SASP component, can prevent the SASP. A three-pronged approach has been adopted to assess the if adalimumab is able to: i) modulate a panel of classic and novel senescence- and SASP-associated markers (interleukin [IL]-6, senescence associated-ß-galactosidase, p16/Ink4a, plasminogen activator inhibitor 1, endothelial nitric oxide synthase, miR-146a-5p/Irak1 and miR-126-3p/Spred1) in human umbilical vein endothelial cells (HUVECs); ii) reduce the paracrine effects of senescent HUVECs' secretome on MCF-7 breast cancer cells, through wound healing and mammosphere assay; and iii) exert significant decrease of miR-146a-5p and increase of miR-126-3p in circulating angiogenic cells (CACs) from psoriasis patients receiving adalimumab in monotherapy.TNF-α blockade associated with adalimumab induced significant reduction in released IL-6 and significant increase in eNOS and miR-126-3p expression levels in long-term HUVEC cultures.A significant reduction in miR-146a-5p expression levels both in long-term HUVEC cultures and in CACs isolated from psoriasis patients was also evident. Interestingly, conditioned medium from senescent HUVECs treated with adalimumab was less consistent than medium from untreated cells in inducing migration- and mammosphere- promoting effects on MCF-7 cells.Our findings suggest that adalimumab can induce epigenetic modifications in cells undergoing senescence, thus contributing to the attenuation of SASP tumor-promoting effects.

MiR-21-5p and miR-126a-3p levels in plasma and circulating angiogenic cells: relationship with type 2 diabetes complications.

Innovative biomarkers are required to manage type 2 diabetic patients (T2DM). We focused our study on miR-126-3p and miR-21-5p levels, as biomarkers of endothelial function and inflammation. MiRNAs levels were measured in plasma from 107 healthy subjects (CTR) and 193 diabetic patients (T2DM), 76 without (T2DM NC) and 117 with (T2DM C) complications. When diabetic complication were analysed as a whole, miR-126-3p and miR-21-5p levels declined significantly from CTR to T2DM NC and T2DM C patients. When miRNAs levels were related to specific complications, significantly higher miR-21-5p levels (0.46 ± 0.44 vs. 0.26 ± 0.33, p < 0.001) and significant lower miR-126-3p levels (0.21 ± 0.21 vs. 0.28 ± 0.22, p = 0.032) were found in T2DM with previous major cardiovascular events (MACE) vs. all the others T2DM patients. To confirm these results we focused on circulating angiogenic cells (CACs) from a subgroup of 10 CTR, 15 T2DM NC and 15 T2DM patients with MACE. CACs from T2DM patients expressed higher miR-21-5p and lower miR-126-3p levels than CACs from CTR. Furthermore, CACs from T2DM + MACE showed the highest levels of miR-21-5p. Circulating miR-21-5p and miR-126-3p emerge as dynamic biomarkers of systemic inflammatory/angiogenic status. Their expression levels in CACs from T2DM with MACE suggest a shift from a proangiogenic to a proinflammatory profile.

Age- and glycemia-related miR-126-3p levels in plasma and endothelial cells.

Circulating miR-126-3p levels were determined in 136 healthy subjects (CTRs) aged 20-90 years and 193 patients with type-2 diabetes mellitus (T2DMs) aged 40-80 years, to explore the combined effect of age and glycemic state on miR-126-3p expression. Moreover, intra/extracellular miR-126-3p levels were measured in human endothelial cells (HUVECs) undergoing senescence under normo/hyper-glycemic conditions. Plasma miR-126-3p was significantly higher in the oldest compared with the youngest CTRs ( <45 vs. >75 years; relative expression: 0.27±0.29 vs. 0.48±0.39, p=0.047). Age-based comparison between CTRs and T2DM demonstrated significantly different miR-126-3p levels only in the oldest (0.48±0.39 vs. 0.22±0.23, p<0.005). After multiple adjustments, miR-126-3p levels were seen to be lower in patients with poor glycemic control, compared with age-matched CTRs. The age-related increase in plasma miR-126-3p found in CTRs was paralleled by a 5/6-fold increase in intra/extracellular miR-126-3p in in vitro-cultured HUVECs undergoing senescence. Notably, significant down- regulation of SPRED-1 protein, a validated miR-126-3p target, was found in senescent HUVECs. Moreover, miR-126-3p expression was down-regulated in intermediate-age HUVECs grown in high-glucose medium until senescence. Aging/senescence-associated miR-126-3p up-regulation is likely a senescence-associated compensatory mechanism that is blunted when endothelial cells are exposed to high glucose levels, a phenomenon that probably occurs in vivo in T2DM patients.