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Recent literature shows that loss of replicative ability and acquisition of a proinflammatory secretory phenotype in senescent cells is coupled with the build-in of nucleic acids in the cytoplasm. Its implication in human age-related diseases is under scrutiny. In human endothelial cells (ECs), we assessed the accumulation of intracellular nucleic acids during in vitro replicative senescence and after exposure to high glucose concentrations, which mimic an in vivo condition of hyperglycemia. We showed that exposure to high glucose induces senescent-like features in ECs, including telomere shortening and proinflammatory cytokine release, coupled with the accrual in the cytoplasm of telomeres, double-stranded DNA and RNA (dsDNA, dsRNA), as well as RNA:DNA hybrid molecules. Senescent ECs showed an activation of the dsRNA sensors RIG-I and MDA5 and of the DNA sensor TLR9, which was not paralleled by the involvement of the canonical (cGAS) and non-canonical (IFI16) activation of the STING pathway. Under high glucose conditions, only a sustained activation of TLR9 was observed. Notably, senescent cells exhibit increased proinflammatory cytokine (IL-1β, IL-6, IL-8) production without a detectable secretion of type I interferon (IFN), a phenomenon that can be explained, at least in part, by the accumulation of methyl-adenosine containing RNAs. At variance, exposure to exogenous nucleic acids enhances both IL-6 and IFN-β1 expression in senescent cells. This study highlights the accrual of cytoplasmic nucleic acids as a marker of senescence-related endothelial dysfunction, that may play a role in dysmetabolic age-related diseases., (© 2024. The Author(s).)

Multicellular tumor spheroids are rapidly emerging as an improved in vitro model with respect to more traditional 2D culturing. Microwell culturing is a simple and accessible method for generating a large number of uniformly sized spheroids, but commercially available systems often do not enable researchers to perform complete culturing and analysis pipelines and the mechanical properties of their culture environment are not commonly matching those of the target tissue. We herein report a simple method to obtain custom-designed self-built microwell arrays made of polydimethylsiloxane or agarose for uniform 3D cell structure generation. Such materials can provide an environment of tunable mechanical flexibility. We developed protocols to culture a variety of cancer and non-cancer cell lines in such devices and to perform molecular and imaging characterizations of the spheroid growth, viability, and response to pharmacological treatments. Hundreds of tumor spheroids grow (in scaffolded or scaffold-free conditions) at homogeneous rates and can be harvested at will. Microscopy imaging can be performed in situ during or at the end of the culture. Fluorescence (confocal) microscopy can be performed after in situ staining while retaining the geographic arrangement of spheroids in the plate wells. This platform can enable statistically robust investigations on cancer biology and screening of drug treatments., (© 2023. The Author(s).)

Methyl-CpG binding protein 2 (MeCP2) is a ubiquitous transcriptional regulator. The study of this protein has been mainly focused on the central nervous system because alterations of its expression are associated with neurological disorders such as Rett syndrome. However, young patients with Rett syndrome also suffer from osteoporosis, suggesting a role of MeCP2 in the differentiation of human bone marrow mesenchymal stromal cells (hBMSCs), the precursors of osteoblasts and adipocytes. Here, we report an in vitro downregulation of MeCP2 in hBMSCs undergoing adipogenic differentiation (AD) and in adipocytes of human and rat bone marrow tissue samples. This modulation does not depend on MeCP2 DNA methylation nor on mRNA levels but on differentially expressed miRNAs during AD. MiRNA profiling revealed that miR-422a and miR-483-5p are upregulated in hBMSC-derived adipocytes compared to their precursors. MiR-483-5p, but not miR-422a, is also up-regulated in hBMSC-derived osteoblasts, suggesting a specific role of the latter in the adipogenic process. Experimental modulation of intracellular levels of miR-422a and miR-483-5p affected MeCP2 expression through direct interaction with its 3' UTR elements, and the adipogenic process. Accordingly, the knockdown of MeCP2 in hBMSCs through MeCP2-targeting shRNA lentiviral vectors increased the levels of adipogenesis-related genes. Finally, since adipocytes released a higher amount of miR-422a in culture medium compared to hBMSCs we analyzed the levels of circulating miR-422a in patients with osteoporosis-a condition characterized by increased marrow adiposity-demonstrating that its levels are negatively correlated with T- and Z-scores. Overall, our findings suggest that miR-422a has a role in hBMSC adipogenesis by downregulating MeCP2 and its circulating levels are associated with bone mass loss in primary osteoporosis., (© 2023. The Author(s).)

Bone marrow mesenchymal stromal cells (BMSCs) are multipotent cells able to self-renew and differentiate, depending on the microenvironment, into adipocytes and osteoblasts. These cells have a limited number of replications and enter replicative senescence during in vitro expansion. The role of DNA methylation (DNAm) assumes importance in cell function and commitment; however, its exact contribution to BMSC differentiation and replicative senescence is still unclear. We performed a genome-wide DNAm analysis on BMSCs cultured in vitro at early passages and induced to differentiate into adipocytes and osteoblasts, and on replicative senescent BMSCs and HUVECs, to identify DNAm patterns of senescence and differentiation. We also compared BMSCs and HUVECs in replicative senescence and found that, in both cellular systems, genome-wide hypomethylation was accompanied by a higher-than-expected overlap of differentially methylated positions (DMPs) and concordance in terms of direction of the change. A Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis on lineage-independent senescence-associated DMPs revealed 16 common pathways, including Insulin resistance, Molecule adhesion, and Wnt/β-catenin signaling. In both adipogenesis and osteogenesis, we observed a general demethylation of CpG sites compared with undifferentiated BMSCs with a higher number of DMPs in osteogenesis. KEGG analysis resulted in 30 pathways enriched in osteoblasts and only 2 in adipocytes when compared to undifferentiated cells. When comparing differentiated BMSCs with senescent ones, osteogenesis exhibited a greater overlap with senescence in terms of number of DMPs and direction of methylation change compared to adipogenesis. In conclusion, this study may be useful for future research on general mechanisms that occur in replicative senescence and furthermore to identify trajectories of BMSC differentiation and common aspects of differentiated and senescent 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.

Pre-eclampsia (PE) is a systemic maternal syndrome affecting 2-8% of pregnancies worldwide and involving poor placental perfusion and impaired blood supply to the foetus. It manifests after the 20 th week of pregnancy as new-onset hypertension and substantial proteinuria and is responsible for severe maternal and newborn morbidity and mortality. Identifying biomarkers that predict PE onset prior to its establishment would critically help treatment and attenuate outcome severity. MicroRNAs are ubiquitous gene expression modulators found in blood and tissues. Trophoblast cell surface antigen (Trop)-2 promotes cell growth and is involved in several cancers. We assessed the PE predictive ability of maternal miR-125b in the first trimester of pregnancy by measuring its plasma levels in women with normal pregnancies and with pregnancies complicated by PE on the 12 th week of gestation. To gain insight into PE pathogenesis we investigated whether Trop-2 is targeted by miR-125b in placental tissue. Data analysis demonstrated a significant association between plasma miR-125b levels and PE, which together with maternal body mass index before pregnancy provided a predictive model with an area under the curve of 0.85 (95% confidence interval, 0.70-1.00). We also found that Trop-2 is a target of miR-125b in placental cells; its localization in the basal part of the syncytiotrophoblast plasma membrane suggests a role for it in the early onset of PE. Altogether, maternal miR-125b proved a promising early biomarker of PE, suggesting that it may be involved in placental development through its action on Trop-2 well before the clinical manifestations of PE., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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.

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., (© 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group on behalf of The International Society for Extracellular Vesicles.)

The postnatal development of nitric oxide (NO)-producing intracallosal neurons was studied in rats by nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry from postnatal day 0 (P0) to P30. NADPH-d-positive neurons (NADPH-d+ Ns ) were detected already at P0, mainly in the rostral region of the corpus callosum (cc). Their location and the intensity of staining allowed them to be classified as type I NO-producing neurons. At P0, tufts of intensely labeled fibers, probably corresponding to the callosal septa described in the monkey and human cc, entered the ventral cc region and reached its dorsal portion. From P5, cell bodies and dendrites were often associated to blood vessels. The number of intracallosal NADPH-d+ Ns rose in the first postnatal days to peak at P5, it declined until P10, and then remained almost constant until P30. Their size increased from P0 to P30, dramatically so (>65%) from P0 to P15. From P10 onward their distribution was adult-like, i.e. NADPH-d+ Ns were more numerous in the lateral and intermediate portions of the cc and diminished close to the midline. In conjunction with previous data, these findings indicate that intracallosal NADPH-d+ Ns could have a role in callosal axon guidance, myelination, refinement processes, and callosal blood flow regulation., (Copyright © 2019. Published by Elsevier B.V.)

Current literature agrees on the notion that efficient DNA repair favors longevity across evolution. The DNA damage response machinery activates inflammation and type I interferon signaling. Both pathways play an acknowledged role in the pathogenesis of a variety of age-related diseases and are expected to be detrimental for human longevity. Here, we report on the anti-inflammatory molecular make-up of centenarian's fibroblasts (low levels of IL-6, type 1 interferon beta, and pro-inflammatory microRNAs), which is coupled with low level of DNA damage (measured by comet assay and histone-2AX activation) and preserved telomere length. In the same cells, high levels of the RNAseH2C enzyme subunit and low amounts of RNAseH2 substrates, i.e. cytoplasmic RNA:DNA hybrids are present. Moreover, RNAseH2C locus is hypo-methylated and RNAseH2C knock-down up-regulates IL-6 and type 1 interferon beta in centenarian's fibroblasts. Interestingly, RNAseH2C locus is hyper-methylated in vitro senescent cells and in tissues from atherosclerotic plaques and breast tumors. Finally, extracellular vesicles from centenarian's cells up-regulate RNAseH2C expression and dampen the pro-inflammatory phenotype of fibroblasts, myeloid, and cancer cells. These data suggest that centenarians are endowed with restrained DNA damage-induced inflammatory response, that may facilitate their escape from the deleterious effects of age-related chronic inflammation.

Parabiosis experiments in mice demonstrated that a young environment could partially rejuvenate multiple tissues of old organisms. However, the circulating mediators responsible of such effect have been elusive so far. Novel results suggest that extracellular vesicles isolated from plasma of young mice increase lifespan in old mice. Here we integrate these findings in a larger framework, advancing the hypothesis that circulating vesicles may mediate the beneficial effect of a young milieu on ageing.

To evaluate the combined effect of age and glycemic state on circulating levels of the inflamma-miR-146a levels, 188 healthy subjects (CTR) aged 20-104 years and 144 type-2 diabetic patients (T2DM), aged 40-80 years, were analyzed. In CTR subjects, miR-146a levels showed a significant age-related decline. When a gender-stratified analysis was ran, the miR-146a age-related trajectory was confirmed only in men and a negative correlation with PAI-1, uric acid, and creatinine was also observed. In women, miR-146a circulating levels showed negative correlations with azotemia, uric acid, waist/hip ratio and ferritin. A significant miR-146a decline with aging was also observed in T2DM patients. Significant positive correlations were found between miR-146a in diabetic patients and total cholesterol, LDL-C, ApoA1, ApoB, and platelets, and negative correlations with serum iron and ferritin. Notably, miR-146a was significantly overexpressed in T2DM patients treated with metformin. MiR-146a levels were significantly lower in diabetic patients than in age-matched CTR and negatively correlated to both fasting glucose and HbA1c in males. Finally, age-related trajectories for circulating miR-146a levels showed an inverted U-shaped relationship; however, in T2DM patients the trajectory was significantly shifted towards lower levels. Our findings support the hypothesis that miR-146a could be a functional biomarker of healthy/unhealthy aging., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Type 2 diabetes mellitus (T2DM) is a metabolic disorder characterized by chronic hyperglycemia associated with alterations in carbohydrate, lipid, and protein metabolism. The prognosis of T2DM patients is highly dependent on the development of complications, and therefore the identification of biomarkers of T2DM progression, with minimally invasive techniques, is a huge need. In the present study, we applied a 1 H-Nuclear Magnetic Resonance ( 1 H-NMR)-based metabolomic approach coupled with multivariate data analysis to identify serum metabolite profiles associated with T2DM development and progression. To perform this, we compared the serum metabolome of non-diabetic subjects, treatment-naïve non-complicated T2DM patients, and T2DM patients with complications in insulin monotherapy. Our analysis revealed a significant reduction of alanine, glutamine, glutamate, leucine, lysine, methionine, tyrosine, and phenylalanine in T2DM patients with respect to non-diabetic subjects. Moreover, isoleucine, leucine, lysine, tyrosine, and valine levels distinguished complicated patients from patients without complications. Overall, the metabolic pathway analysis suggested that branched-chain amino acid (BCAA) metabolism is significantly compromised in T2DM patients with complications, while perturbation in the metabolism of gluconeogenic amino acids other than BCAAs characterizes both early and advanced T2DM stages. In conclusion, we identified a metabolic serum signature associated with T2DM stages. These data could be integrated with clinical characteristics to build a composite T2DM/complications risk score to be validated in a prospective cohort., Competing Interests: The authors declare no conflict of interest.

The receptor for advanced glycation end-products (RAGE) recognizes several ligands involved in inflammatory diseases. Two circulating soluble isoforms exist: esRAGE derived from alternative splicing and cRAGE generated by the membrane-bound RAGE (FL-RAGE) proteolysis. Together, esRAGE and cRAGE constitute sRAGE and function as decoy receptors preventing FL-RAGE/ligands binding.We determined serum concentration of both, esRAGE and cRAGE, and their ligands AGEs, HMGB1 and S100A8/A9 in a healthy population of 169 subjects aged 20-90 years. cRAGE showed a negative (r=-0.375, P<0.0001) while AGEs (r=0.160, P=0.0384) and S100A8/A9 (r=0.207, P=0.0091) a positive correlation with age. esRAGE did not change during aging and inversely correlated with Hemoglobin, ALT, insulin, HOMA index, Waist-Hip ratio (W/H), Waist Circumference (WC) and positively with AGEs. cRAGE exhibited also an inverse correlation with WC, W/H, PAI-1, HMGB1, AGEs and S100A8/A9. Age, W/H, HMGB1, S100A8/A9 and AGEs are independent predictors of cRAGE, whereas W/H and AGEs associate with esRAGE. Treatment of cells with glycated albumin reduced cRAGE production and upregulated FL-RAGE.These results indicate that in a healthy population cRAGE is a biomarker of aging while esRAGE represents a more reliable marker of obesity and insulin resistance. Hence, sRAGE isoforms levels could be differentially associated with age-related diseases risk factors.

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 approaches., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Metformin is the first-choice therapy to lower glycaemia and manage type 2 diabetes. Continuously emerging epidemiological data and experimental models are showing additional protective effects of metformin against a number of age-related diseases (ARDs), e.g., cardiovascular diseases and cancer. This evidence has prompted the design of a specific trial, i.e., the Targeting Aging with Metformin (TAME) trial, to test metformin as an anti-ageing molecule. However, a unifying or prevailing mechanism of action of metformin is still debated. Here, we summarize the epidemiological data linking metformin to ARD prevention. Then, we dissect the deeply studied mechanisms of action explaining its antihyperglycemic effect and the putative mechanisms supporting its anti-ageing properties, focusing on studies using clinically pertinent doses. We hypothesize that the molecular observations obtained in different models with metformin could be indirectly mediated by its effect on gut flora. Novel evidence suggests that metformin reshapes the human microbiota, promoting the growth of beneficial bacterial species and counteracting the expansion of detrimental bacterial species. In turn, this action would influence the balance between pro- and anti-inflammatory circulating factors, thereby promoting glycaemic control and healthy ageing. This framework may reconcile diverse observations, providing information for designing further studies to elucidate the complex interplay between metformin and the metabiome harboured in mammalian body compartments, thereby paving the way for innovative, bacterial-based therapeutics to manage type 2 diabetes and foster a longer healthspan., (Copyright © 2018 Elsevier B.V. All rights reserved.)

During senescence, cells undergo distinctive biochemical and morphological changes and become dysfunctional. MiRNAs are involved in the senescence process and specific miRNAs can localize to mitochondria (mitomiRs). We hypothesized that part of the typical alterations of senescence may depends on mitomiRs deregulation. Therefore, we thoroughly explored the phenotype of human endothelial cells undergoing replicative senescence (sHUVECs) and observed elongated/branched mitochondria, accumulation of autophagic vacuoles (AVs), increased ROS and IL-1β production and reduced expression of Bcl-2 compared to younger cells (yHUVECs). Despite these pro-apoptotic features, sHUVECs are more resistant to serum deprivation, conceivably due to development of pro-survival strategies such as upregulation of Bcl-xL and Survivin. We demonstrate that mitomiR-181a, -34a, and -146a, are overexpressed and localize to mitochondria in sHUVECs compared with yHUVECs and that they: i) down-regulate Bcl-2, ii) induce permeability transition pore opening and activation of caspase-1 and 3, iii) affect sensitivity to apoptosis and iv) promote the conversion of LC3-I to LC3-II. Overall, we document for the first time that some mitomiRs can act as mediators of the multiple but functionally linked biochemical and morphological changes that characterize aging cells and that they can promote different cellular outcomes according to the senescence status of the cell.

Specific microRNAs (miRs), including the "angio-miR-126" and the "inflamma-miR-146a-5p," have been proposed as biomarkers and even therapeutic targets of obesity-associated metabolic diseases. Physical activity, a key measure of prevention for obesity and its complications, is reported to influence the expression of these miRs. In this study, we investigate whether a physical activity program proven to improve metabolic parameters in obese patients can correct the circulating levels of these miRs. Plasma miR-126 and miR-146a-5p were measured in a cohort of obese patients (n = 31, 16F + 15M) before and after the 3-month physical activity program of the CURIAMO trial (registration number for clinical trials: ACTRN12611000255987) and in 37 lean controls (24F + 13M). miR-146a-5p, but not miR-126, was significantly increased in obese patients as compared with lean controls and decreased in approximately two-thirds of the participants post-intervention with a response that positively correlated with pre-intervention levels of this miR. Waist circumference, the inflammatory cytokine IL-8 and lipid parameters, principally total cholesterol, showed the strongest correlation with both the baseline levels and post-intervention correction of miR-146a-5p. Post-hoc analysis of experimental data supports the use of miR-146a-5p as a biomarker and predictor of the clinical response to physical activity in obese patients. Furthermore, miR-146a-5p expression was confirmed to increase together with that of the inflammatory genes TLR4, NF-κB, IL-6, and TNF-α in LPS-stimulated human mononuclear leukocytes. In conclusion, the inflamma-miR-146a-5p can serve as a personalized predictor of clinical outcome in obese patients entering physical activity weight-reduction programs. © 2018 IUBMB Life, 70(10):1012-1022, 2018., (© 2018 International Union of Biochemistry and Molecular Biology.)

In bone marrow (BM), hematopoietic elements are mingled with adipocytes (BM-A), which are the most abundant stromal component in the niche. BM-A progressively increase with aging, eventually occupying up to 50% of BM cavities. In this work, the role played by BM-A was explored by studying primary human BM-A isolated from hip surgery patients at the molecular level, through microarray analysis, and at the functional level, by assessing their relationship with primary human hematopoietic stem cells (HSC) by the long-term culture initiating cell (LTC-IC) assay. Findings demonstrated that BM-A are capable of supporting HSC survival in the LTC-IC assay, since after 5 weeks of co-culture, HSC were still able to proliferate and differentiate. Furthermore, critical molecules such as C-X-C motif chemokine 12 (CXCL12), interleukin (IL)-8, colony-stimulating factor 3 (CSF3), and leukaemia inhibitory factor (LIF), were expressed at similar levels in BM-A and in primary human BM mesenchymal stromal cells (BM-MSC), whereas IL-3 was higher in BM-A. Interestingly, BM-A displayed a different gene expression profile compared with subcutaneous adipose tissue adipocytes (AT-A) collected from abdominal surgery patients, especially in terms of regulation of lipid metabolism, stemness genes, and white-to-brown differentiation pathways. Accordingly, analysis of the gene pathways involved in hematopoiesis regulation showed that BM-A are more closely related to BM-MSC than to AT-A. The present data suggest that BM-A play a supporting role in the hematopoietic niche and directly sustain HSC survival., (© 2017 Wiley Periodicals, Inc.)

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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Exosomes are nanovesicles formed by inward budding of endosomal membranes. They exert complex immunomodulatory effects on target cells, acting both as antigen-presenting vesicles and as shuttles for packets of information such as proteins, coding and non-coding RNA, and nuclear and mitochondrial DNA fragments. Albeit different, all such functions seem to be encompassed in the adaptive mechanism mediating the complex interactions of the organism with a variety of stressors, providing both for defense and for the evolution of symbiotic relationships with others organisms (gut microbiota, bacteria, and viruses). Intriguingly, the newly deciphered human virome and exosome biogenesis seem to share some physical-chemical characteristics and molecular mechanisms. Exosomes are involved in immune system recognition of self from non-self throughout life: they are therefore ideal candidate to modulate inflamm-aging, the chronic, systemic, age-related pro-inflammatory status, which influence the development/progression of the most common age-related diseases (ARDs). Not surprisingly, recent evidence has documented exosomal alteration during aging and in association with ARDs, even though data in this field are still limited. Here, we review current knowledge on exosome-based trafficking between immune cells and self/non-self cells (i.e. the virome), sketching a nano-perspective on inflamm-aging and on the mechanisms involved in health maintenance throughout life., (Copyright © 2017 Elsevier B.V. All rights reserved.)

β-Galactosidase (β-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 β-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 β-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 β-Gal activity in young and senescent endothelial cells (HUVECs) cultured in normo- and hyper-glycaemic environment. A significant age-related increase of plasmatic β-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. β-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 β-Gal activity in milieu compared to normo-glycaemic condition. Overall our data reinforce the notion that plasmatic β-Gal activity could be a systemic biomarker of aging, whereas T2DM patients are characterized by a different age-releated trend., Competing Interests: CONFLICTS OF INTEREST None of the authors has competing interests.

Introduction: The expression of substance P (SP) receptor (neurokinin 1, NK1) was studied in the rat corpus callosum (cc) from postnatal day 0 (the first 24 hr from birth, P0) to P30., Methods: We used immunocytochemistry to study the presence of intracallosal NK1-immunopositive neurons (NK1 IP -n ) during cc development., Results: NK1 IP -n first appeared on P5. Their number increased significantly between P5 and P10, it remained almost constant between P10 and P15, then declined slightly until P30. The size of intracallosal NK1 IP -n increased constantly from P5 (102.3 μm 2 ) to P30 (262.07 μm 2 ). From P5 onward, their distribution pattern was adult-like, that is, they were more numerous in the lateral and intermediate parts of the cc, and declined to few or none approaching the midline. At P5, intracallosal NK1 IP -n had a predominantly round cell bodies with primary dendrites of different thickness from which originated thinner secondary branches. Between P10 and P15, dendrites were longer and more thickly branched, and displayed several varicosities as well as short, thin appendages. Between P20 and P30, NK1 IP -n were qualitatively indistinguishable from those of adult animals and could be classified as bipolar (fusiform and rectangular), round-polygonal, and pyramidal (triangular-pyriform)., Conclusions: Number of NK1 IP -n increase between P5 and P10, then declines, but unlike other intracallosal neurons, NK1 IP -n make up a significant population in the adult cc. These findings suggest that NK1 IP -n may be involved in the myelination of callosal axons, could play an important role in their pathfinding. Since they are also found in adult rat cc, it is likely that their role changes during lifetime.

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., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Leukocyte telomere length (LTL) shortening is found in a number of age-related diseases, including type 2 diabetes (T2DM). In this study its possible association with mortality was analyzed in a sample of 568 T2DM patients (mean age 65.9 ± 9 years), who were followed for a median of 10.2 years (interquartile range 2.2). A number of demographic, laboratory and clinical parameters determined at baseline were evaluated as mortality risk factors. LTL was measured by quantitative real-time PCR and reported as T/S (telomere-to-single copy gene ratio). Age, gender, creatinine, diabetes duration at baseline, and LTL were significantly different between T2DM patients who were dead and alive at follow-up. In the Cox regression analysis adjusted for the confounding variables, shorter LTL, older age, and longer disease duration significantly increased the risk of all-cause mortality (HR = 3.45, 95%CI 1.02-12.5, p = 0.004). Kaplan-Maier analysis also found a different cumulative mortality risk for patients having an LTL shorter than the median (T/S ≤0.04) and disease duration longer than the median (>10 years) (log-rank = 11.02, p = 0.011). Time-dependent mortality risk stratification showed that T2DM duration and LTL combined was a fairly good predictor of mortality over the first 76 months of follow-up.In conclusion, LTL combined with clinical parameters can provide additive prognostic information on mortality risk in T2DM patients., Competing Interests: None of the authors have any competing interests.

Electrical stimulation of the dorsal columns (DCs; spinal cord stimulation; SCS) has been proposed to treat chronic neuropathic pain. SCS may activate a dual mechanism that would affect both the spinal cord and supraspinal levels. Stimulation of DCs or DC nuclei (DCN) in animals where neuropathic pain has been induced causes activation of brainstem centers including the periaqueductal gray (PAG), which is involved in the endogenous pain suppression system. Biotinylated dextran-amine (BDA) was iontophoretically injected into the DCN to analyze the ascending projection directed to the PAG. Separate injections into the gracile nucleus (GrN) and the cuneate nucleus (CunN) showed BDA-positive fibers terminating in different regions of the contralateral PAG. GrN-PAG afferents terminated in the caudal and middle portions of PAG-l, whereas CunN-PAG fibers terminated in the middle and rostral portions of PAG-l. Based on the DCN somatotopic map, the GrN sends information to the PAG from the contralateral hindlimb and the tail and the CunN from the contralateral forelimb, shoulder, neck and ear. This somatotopic organization is consistent with earlier electrophysiological and PAG stimulation studies. These fibers could form part of the DCs-brainstem-spinal cord loop, which may be involved in the inhibitory effects of SCS on neuropathic pain., (Copyright © 2016. Published by Elsevier Ireland Ltd.)

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.

The corpus callosum (cc) contains nitric oxide (NO)-producing neurons. Because NO is a potent vasodilator, these neurons could translate neuronal signals into vascular responses that can be detected by functional brain imaging. Substance P (SP), one of the most widely expressed peptides in the CNS, also produces vasomotor responses by inducing calcium release from intracellular stores through its preferred neurokinin 1 (NK1) receptor, thus inducing NO production via activation of neuronal NO synthase (nNOS). Single- and double-labeling experiments were performed to establish whether NK1-immunopositive neurons (NK1IP -n) are found in the rat cc and the extent of NK1 colocalization with nNOS. NK1IP -n were seen to constitute a large neuronal population in the cc and had a distribution similar to that of nNOSIP neurons (nNOSIP -n). NK1IP -n were numerous in the lateral cc and gradually decreased in the more medial portions, where they were few or absent. Intracallosal NK1IP -n and their dendritic trees were intensely labeled, allowing classification into four morphological types: bipolar, round, polygonal, and pyramidal. Confocal microscopic examination demonstrated that nearly all NK1IP -n contained nNOS (96.43%) and that 84.59% of nNOSIP -n co-expressed NK1. These data suggest that the majority of intracallosal neurons can release NO as a result of the action of SP. A small proportion of nNOSIP -n does not contain NK1 and is not activated by SP; these neurons may release NO via alternative mechanisms. The possible mechanisms by which intracallosal neurons release NO are also reviewed., (© 2014 Wiley Periodicals, Inc.)

Introduction: The aim of this study was to determine the presence and distribution of nitric oxide (NO)-producing neurons in the rat corpus callosum (cc)., Material and Methods: To investigate this aspect of cc organization we used nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry and neuronal NO synthase (nNOS) immunocytochemistry., Results: Intense NADPH-d-positive (NADPH-d+) neurons were found along the rostrocaudal extension of the cc (sagittal sections). They were more numerous in the lateral cc and gradually decreased in the more medial regions, where they were very few or absent. The Golgi-like appearance of NADPH-d+ intracallosal neurons allowed dividing them into five morphological types: (1) bipolar; (2) fusiform; (3) round; (4) polygonal; and (5) pyramidal. The number of NADPH-d+ neurons (both hemispheres) was counted in two brains using 50-μm thick sections. In the first brain, counts involved 145 sections and neurons were 2959; in the second, 2227 neurons were counted in 130 sections. The distribution and morphology of nNOS-immunopositive (nNOSIP) neurons was identical to that of NADPH-d+neurons. Some of these neurons were observed in the cc ependymal region, where they might be in contact with cerebrospinal fluid (CSF), monitoring its composition, pH, and osmolality changes, or playing a role in regulating the synthesis and release of several peptides. The somatic, dendritic, and axonal processes of many NADPH-d+/nNOSIP neurons were closely associated with intracallosal blood vessels., Conclusions: Such close relationship raises the possibility that these neurons are a major source of NO during neural activity. As NO is a potent vasodilator, these findings strongly suggest that NO-positive neurons transduce neuronal signals into vascular responses in selected cc regions, thus giving rise to hemodynamic changes detectable by neuroimaging.

The distribution, colocalization with enzymes producing nitric oxide (NO), and the synaptic organization of neurons containing two calcium-binding proteins (CaBPs) - parvalbumin (Parv) and calbindin-D28K (Calb) - were investigated in the rat periaqueductal gray matter (PAG). Parv-immunopositive (ParvIP) neurons were detected in the mesencephalic nucleus and rarely in the PAG. CalbIP neurons were found both in the dorsolateral (PAG-dl) and ventrolateral PAG (PAG-vl); their size ranged from 112.96 μm(2) (PAG-dl) to 125.13 μm(2) (PAG-vl). Ultrastructurally Parv and Calb immunoreactivity was mostly found in dendritic profiles. Axon terminals containing each of the two CaBPs formed symmetric synapses. Moreover both Parv and Calb were used to label a subpopulation of NO-producing neurons. Colocalization was investigated using two protocols: (i) a combination of Calb and Parv immunocytochemistry (Icc) with nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry (Hi) and (ii) neuronal NO synthase-Icc (nNOS) (immunofluorescence). Both techniques demonstrated a complete lack of colocalization of Parv and NADPH-d/nNOS in PAG neurons. Double-labeled (DL) neurons (Calb-NADPH-d; Calb-nNOS) were detected in PAG-dl. NADPH-d-Hi/Calb-Icc indicated that 41-47% of NADPH-d-positive neurons contained Calb, whereas 17-23% of CalbIP cells contained NADPH-d. Two-color immunofluorescence revealed that 53-66% of nNOSIP cells colocalized with Calb and 24-34% of CalbIP neurons contained nNOS. DL neuron size was 104.44 μm(2); neurons labeled only with NADPH-d or Calb measured 89.793 μm(2) and 113.48 μm(2), respectively. Together with previous findings (Barbaresi et al. [2012]) these data suggest that: Therefore the important aspect of the PAG intrinsic organization emerging from this and previous double-labeling studies is the chemical diversity of NO-synthesizing neurons, which is likely related to the different functions in which these neurons are involved., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The pattern of distribution and colocalization of the calcium-binding protein calretinin (Cal) and of enzymes producing nitric oxide (NO) was examined in the rat periaqueductal gray matter (PAG) using two different experimental approaches, by combining Cal immunocytochemistry with NADPH-diaphorase (NADPH-d) histochemistry and with NOS immunocytochemistry, respectively. Cal-immunopositive neurons were found throughout the rostrocaudal extension of both dorsolateral (PAG-dl) and ventrolateral PAG (PAG-vl). Double-labeled neurons were found only in PAG-dl. The first experimental approach indicated that 33-41% of the NADPH-d-positive (Nadph+) cells were immunoreactive for Cal, whereas NADPH-d activity appeared in 19-26% of the Cal-immunopositive (Cal(IP) ) neurons. Two-color immunofluorescence revealed that ∼39-43% of NOS-immunoreactive (NOS(IR) ) neurons were double-labeled with Cal and ∼23% of Cal(IP) neurons expressed NOS immunoreactivity. Measurement in semithin sections of the size of the three neuronal populations found in PAG-dl, showed that Cal(IP) neurons had a cross-sectional area of 94.7 μm², whereas Nadph+ neurons and double-labeled neurons were slightly smaller, having a cross-sectional area of 90.5 and 91.4 μm², respectively. On electron microscopy, Cal(IP) axon terminals formed either symmetric or asymmetric synapses; although the latter synapses were more numerous, both types contacted preferentially Cal(IP) dendrites. These experiments suggest that PAG-dl is characterized by a high degree of heterogeneity., (Copyright © 2011 Wiley Periodicals, Inc.)