Alterations in the Genomic Distribution of 5hmC in In Vivo Aged Human Skin Fibroblasts.

5-Hydroxymethylcytosine (5hmC) is a functionally active epigenetic modification. We analyzed whether changes in DNA 5-hydroxymethylation are an element of age-related epigenetic drift. We tested primary fibroblast cultures originating from individuals aged 22-35 years and 74-94 years. Global quantities of methylation-related DNA modifications were estimated by the dot blot and colorimetric methods. Regions of the genome differentially hydroxymethylated with age (DHMRs) were identified by hMeDIP-seq and the MEDIPS and DiffBind algorithms. Global levels of DNA modifications were not associated with age. We identified numerous DHMRs that were enriched within introns and intergenic regions and most commonly associated with the H3K4me1 histone mark, promoter-flanking regions, and CCCTC-binding factor (CTCF) binding sites. However, only seven DHMRs were identified by both algorithms and all of their settings. Among them, hypo-hydroxymethylated DHMR in the intron of Rab Escort Protein 1 (CHM) coexisted with increased expression in old cells, while increased 5-hydroxymethylation in the bodies of Arginine and Serine Rich Protein 1 (RSRP1) and Mitochondrial Poly(A) Polymerase (MTPAP) did not change their expression. These age-related differences were not associated with changes in the expression of any of the ten-eleven translocation (TET) enzymes or their activity. In conclusion, the distribution of 5hmC in DNA of in vivo aged human fibroblasts underwent age-associated modifications. The identified DHMRs are, likely, marker changes.

Age-related epigenetic drift deregulates SIRT6 expression and affects its downstream genes in human peripheral blood mononuclear cells.

Sirtuin 6 (SIRT6) exerts a protective effect on health and extends the lives of model organisms. We, therefore, aimed to clarify whether age-related epigenetic drift is responsible for differences in SIRT6 expression in peripheral blood mononuclear cells (PBMCs) of healthy young (n = 55, mean age 27.5 ± 4.4 years), middle-aged (n = 51, 65.4 ± 3.3 years), and long-lived (n = 51, 93.9 ± 3.6 years) humans. In silico analysis was performed using the STRING network. No age-related differences were observed in the percentage of SIRT6 CpG island methylation. However, the age affected the expression of miR-34a-5p, miR-125a-5p, miR-186-5p, miR-342-5p and miR-766-3p (all p < 0.0001), miR-181-2-3p and Let-7c (both p = 0.0003), and miR-103a-3p (p = 0.0069). A negative association was observed between SIRT6 mRNA and miR-186-5p (rs = -0.25, p = 0.026), and a positive association was observed with miR-34a-5p (rs = 0.31, p = 0.0055) and miR-181a-2-3p (rs = 0.39, p = 0.0002). SIRT6 mRNA also negatively correlated with the expression of TP53 (rs = -0.41, p = 0.0126) and MYC (rs = -0.35, p = 0.0448). Notably, the expression of several miRNAs and genes was similar in young and long-lived groups but different from the middle-aged group. We conclude that age-related epigenetic changes can affect the expression of SIRT6 in PBMCs and, in this way, possibly influence immunosenescence. Moreover, molecular events could differentiate 'normal' ageing from that of long-lived individuals.

Alterations in 5hmC level and genomic distribution in aging-related epigenetic drift in human adipose stem cells.

Aim: To clarify mechanisms affecting the level and distribution of 5-hydroxymethylcytosine (5hmC) during aging. Materials & methods: We examined levels and genomic distribution of 5hmC along with the expression of ten-eleven translocation methylcytosine dioxygenases (TETs) in adipose stem cells in young and age-advanced individuals. Results: 5hmC levels were higher in adipose stem cells of age-advanced than young individuals (p = 0.0003), but were not associated with age-related changes in expression of TETs. 5hmC levels correlated with population doubling time (r = 0.62; p = 0.01). We identified 58 differentially hydroxymethylated regions. Hypo-hydroxymethylated differentially hydroxymethylated regions were approximately twofold enriched in CCCTC-binding factor binding sites. Conclusion: Accumulation of 5hmC in aged cells can result from inefficient active demethylation due to altered TETs activity and reduced passive demethylation due to slower proliferation.

Acquired immunological imbalance after surgery with cardiopulmonary bypass due to epigenetic over-activation of PU.1/M-CSF.

BACKGROUND: It has been shown that severe insult to the immune system may trigger prolonged macrophage characteristics associated with excessive release of monocyte colony stimulating factor (M-CSF). However, it is unclear how persistent is the macrophage-like characteristics in circulating monocytes (MO). In this study, 20 patients who underwent non-emergent cardiopulmonary bypass had their monocytes characterized before surgery and 3 months after surgery. METHODS: We assessed the macrophage characteristics of MO using cytokine production, surface marker expression, an ability to stimulate T cells, and methylation of the promoter region of the gene encoding PU.1, a critical component to M-CSF production. MO function as well as activation and differentiation potential were longitudinally assessed. RESULTS: At 3 months after cardiopulmonary bypass, monocytes exhibited increased expression of MRP8, transforming growth factor-ß/latency-associated peptide, suppressor of cytokine signaling 3 while phagocytic properties were increased. Concomitantly, we observed a decreased expression of CD86, a decreased ability to form regulatory dendritic cells, and a diminished ability to stimulate T cells. These characteristics were accompanied by a persistent increase in the secretion of M-CSF, over-activation of PU.1, and decreased methylation of the PU.1 promoter region. Serum levels of C-reactive protein and anti-cytomegalovirus IgG antibody titers were also elevated in some patients at 3 months after surgery. CONCLUSIONS: We concluded that at 3 months after cardiopulmonary bypass, monocytes continued to express a new macrophage-like milieu that was associated with the persistent activation of the PU.1/M-CSF pathway.

miR-34a and miR-9 are overexpressed and SIRT genes are downregulated in peripheral blood mononuclear cells of aging humans.

Increased expression of sirtuins lowers the risk of age-related diseases, while their role in the regulation of longevity is not firmly established. Since aging is associated with immunosenescence, we tested whether sirtuin expression was modified in peripheral blood mononuclear cells (PBMC) in an age-related manner and whether this might result from altered expression of the selected miRNAs. The expression of seven SIRT genes and of SIRT1 mRNA-interacting miR-9, miR-34a, miR-132, and miR-199a-5p was evaluated by real-time PCR in PBMC originating from young (Y, n = 57, mean age 27 ± 4.3 years), elderly (E, n = 52, 65 ± 3.4 years), and long-lived (L, n = 56, 94 ± 3.5 years) individuals. Older age was associated with a decreased expression of the majority of the SIRT genes. Most severely affected were median expressions of SIRT1 ( P = 0.000001 for the whole studied group, Y vs. E: P < 0.000001, Y vs. L: P < 0.000001), and of SIRT3 ( P = 0.000001, Y vs. E: P = 0.000004, Y vs. L: P = 0.000028). Older age was also associated with the increased median expression of miR-34a ( P = 0.000001, Y vs. E: P = 0.001, Y vs. L: P = 0.000004) and of miR-9 ( P = 0.05, Y vs. L: P = 0.054). In functional studies, miR-9 interacted with the 3'UTR of SIRT1 mRNA. The SIRT1 mRNA level negatively correlated with the expression of miR-34a ( r = -0.234, P = 0.003). In conclusion, age-related decrease of SIRT1 expression in PBMC might in part result from overexpression of miR-34a and miR-9. In addition, the sustained expression of the SIRT genes in PBMC is not a prerequisite to longevity in humans but might be one of the reasons for the immune system dysfunction in the elderly. Impact statement High expression of sirtuins, particularly SIRT1, lowers the risk of age-related diseases and probably slows down the rate of aging; therefore, their sustained expression should be one of the features of longevity. However, in this work we show that in peripheral blood mononuclear cells (PBMC) of long-lived individuals, expression of majority of the SIRT genes is significantly lower than in cells of young study subjects. In long-lived individuals, downregulation of SIRT1 coexists with upregulation of SIRT1 mRNA-interacting miR-34a and miR-9, indicating the role of epigenetic drift in age-dependent deregulation of SIRT1 expression. Such constellation of SIRT1, miR-34a, and miR-9 expression in PBMC of successfully aging long-lived individuals indicates that, at least in these individuals, it is not a risk factor for morbidity and mortality. It might however affect the function of the immune system and, therefore, aging individuals can profit from interventions increasing the level of SIRT1.

Long-term Monocyte Dysfunction after Sepsis in Humanized Mice Is Related to Persisted Activation of Macrophage-Colony Stimulation Factor (M-CSF) and Demethylation of PU.1, and It Can Be Reversed by Blocking M-CSF In Vitro or by Transplanting Naïve Autologous Stem Cells In Vivo.

The duration of post-sepsis long-term immune suppression is poorly understood. Here, we focused on the role of monocytes (MO) as the pivotal cells for long-term regulation of post-sepsis milieu. Lost ability of MO to adapt is seen in several acute conditions, but it is unclear for how long MO aberrancy post-sepsis can persist. Interestingly, the positive feedback loop sustaining secretion of macrophage-colony stimulation factor (M-CSF) can persist even after resolution of sepsis and significantly alters performance of MO. Here, we investigated the activation of M-CSF, and it as critical regulator of PU.1 in mice surviving 28 days after sepsis. Our primary readout was the ability of MO to differentiate into dendritic cells (DCs; MO→iDC) in vitro since this is one of the critical processes regulating a successful transition from innate to acquired immunity. We utilized a survival modification of the cecal ligation and puncture (CLP) model of sepsis in humanized mice. Animals were sacrificed 28 days after CLP (tCLP+28d). Untouched (CONTR) or sham-operated (SHAM) animals served as controls. Some animals received rescue from stem cells originally used for grafting 2 weeks after CLP. We found profound decrease of MO→iDC in the humanized mice 28 days after sepsis, demonstrated by depressed expression of CD1a, CD83, and CD209, diminished production of IL-12p70, and depressed ability to stimulate T cells in mice after CLP as compared to SHAM or CONTR. In vitro defect in MO→iDC was accompanied by in vivo decrease of BDCA-3+ endogenous circulating DC. Interestingly, post-CLP MO had persistent activation of M-CSF pathway, shown by exaggerated secretion of M-CSF, activation of PU.1, and demethylation of SPII. Neutralization of the M-CSF in vitro reversed the post-CLP MO→iDC aberration. Furthermore, transplantation of naïve, autologous stem cell-derived MO restored CLP-deteriorated ability of MO to become DC, measured as recovery of CD1a expression, enhanced production of IL-12p70, and ability of IL-4 and GM-CSF MO to stimulate allogeneic T cells. Our results suggest the role of epigenetic mediated M-CSF aberration in mediating post-sepsis immune system recovery.

Age-related changes of leptin and leptin receptor variants in healthy elderly and long-lived adults.

AIM: Aging is usually associated with hyperleptinemia and leptin resistance, both increasing the risk of age-related diseases. It was relevant to establish if healthily aging, non-obese individuals develop changes in leptin, the soluble leptin receptor (OB-Re), free leptin index (FLI), in methylation of the leptin receptor gene (LEPR) promoter, and in the expression of long (OB-Rb) and short (OB-Ra) leptin receptor isoforms. METHODS: We analyzed these parameters in 38 young (aged 26.8 ± 3.6 years), 37 elderly (aged 64.7 ± 3.1 years) and 39 long-lived (aged 94.2 ± 3.7 years) healthy, non-obese Polish Caucasians. RESULTS: In elderly men, the median concentration of leptin and the median FLI were significantly higher than in young men (P = 0.009 and P = 0.007, respectively), which was probably partly due to a higher mean body mass index of the elderly study participants. In peripheral blood mononuclear cells, the expression of functionally active OB-Rb did not depend on age or sex, whereas the expression of OB-Ra was lower in the elderly and long-lived groups than in the young group (P < 0.0001 and P = 0.002, respectively), mostly due to changes observed in women. Most likely, this age-related decrease was not due to hypermethylation of the LEPR promoter, as methylation of the +20 to +281 fragment of the CpG island did not change with age. CONCLUSIONS: In healthy, non-obese individuals, only some elements of the leptin axis slightly change with age. On that basis, we suggest that proper function of this axis might be required for this particular phenotype of aging. The present results should, however, be replicated in prospective studies and in other ethnic groups.

Functional polymorphisms of the leptin and leptin receptor genes are associated with longevity and with the risk of myocardial infarction and of type 2 diabetes mellitus.

INTRODUCTION: Longevity is commonly associated with good health and with delayed onset of age-related diseases with usually benign course. Leptin (LEP) significantly affects metabolism and numerous functions of the organism. To find out if extreme longevity and its phenotype are associated with genetic variants of leptin and leptin receptor (LEPR) genes, we analysed the frequencies of the -2548 G/A and +19 G/A LEP, as well as the K109R, Q223R, and K656N LEPR polymorphisms in centenarians and in control groups. MATERIAL AND METHODS: The frequencies of the LEP and LEPR polymorphisms were tested by restriction fragment length polymorphism in 128 centenarians, 414 young controls (Y), 226 myocardial infarction (MI) patients, and 190 type 2 diabetes mellitus (DM2) patients. RESULTS: The GG genotype of the -2548 G/A LEP polymorphism was significantly more common in centenarians than in the Y, MI and DM2 groups (p = 0.048, p = 0.003, p = 0.049, respectively). In addition, the AA genotype of the K109R LEPR polymorphism was significantly less frequent in centenarians than in the Y, MI, and DM2 groups (p = 0.026, p = 0.013, and p = 0.001, respectively). CONCLUSIONS: We suggest that the leptin pathway plays a role in the regulation of longevity, possibly by modulating the risk of development of MI and of DM2.

Small-molecule hormones: molecular mechanisms of action.

Small-molecule hormones play crucial roles in the development and in the maintenance of an adult mammalian organism. On the molecular level, they regulate a plethora of biological pathways. Part of their actions depends on their transcription-regulating properties, exerted by highly specific nuclear receptors which are hormone-dependent transcription factors. Nuclear hormone receptors interact with coactivators, corepressors, basal transcription factors, and other transcription factors in order to modulate the activity of target genes in a manner that is dependent on tissue, age and developmental and pathophysiological states. The biological effect of this mechanism becomes apparent not earlier than 30-60 minutes after hormonal stimulus. In addition, small-molecule hormones modify the function of the cell by a number of nongenomic mechanisms, involving interaction with proteins localized in the plasma membrane, in the cytoplasm, as well as with proteins localized in other cellular membranes and in nonnuclear cellular compartments. The identity of such proteins is still under investigation; however, it seems that extranuclear fractions of nuclear hormone receptors commonly serve this function. A direct interaction of small-molecule hormones with membrane phospholipids and with mRNA is also postulated. In these mechanisms, the reaction to hormonal stimulus appears within seconds or minutes.

Total and high molecular weight adiponectin and level-modifying polymorphisms of ADIPOQ in centenarians.

INTRODUCTION: Adiponectin demonstrates a protective role against the development of obesity, type 2 diabetes mellitus, and cardiovascular disease. The -11377C 〉 G, -11391G 〉 A, and -11426A 〉 G promoter polymorphisms of ADIPOQ gene influence the level of circulating adiponectin. We examined the level of total and high molecular weight (HMW) adiponectin in centenarians and associated it with biochemical parameters. We checked if the expression and concentration-modifying polymorphisms of ADIPOQ are associated with extreme longevity. MATERIAL AND METHODS: Total and HMW adiponectin were examined using ELISA in 40 female centenarians. The frequencies of the ADIPOQ polymorphisms were tested by restriction fragment length polymorphism in 148 centenarians, 414 young controls, in 207 myocardial infarction patients, and in 190 type 2 diabetes mellitus patients. RESULTS: The mean concentration of total adiponectin in centenarians was 13.19 ± 1.37 mg/mL and of HMW adiponectin it was 9.17 ± 1.15 mg/mL. They were positively correlated with HDL (r = 0.4696, p = 0.0025 and r = 0.3912, p = 0.015, respectively), and negatively with BMI (r = -0.3702, p = 0.034 and r = -0.3963, p = 0.025) and triglycerides (r = -0.346, p = 0.028 and r = -0.3227, p = 0.045). A very rare AA genotype of the -11391G 〉 A polymorphism was significantly more common in centenarians than in young controls (p = 0.026) and, while compared to the GG genotype, it was associated with a 2.4-fold higher mean concentration of total adiponectin (26.53 ± 13.29 mg/ mL v. 10.97 ± 4.28 mg/mL) and with an almost 3-fold higher mean HMW adiponectin (20.65 ± 12.72 mg/mL v. 7.36 ± 3.35 mg/mL). CONCLUSIONS: Serum adiponectin concentration in female centenarians is associated with biochemical parameters that are favourable for cardiovascular risk. We suggest that adiponectin might be of importance for extreme longevity.

Aging is accompanied by a progressive decrease of expression of the WRN gene in human blood mononuclear cells.

The WRN gene encodes DNA helicase participating in genome maintenance. We looked for associations of natural aging with expression and methylation of this gene in blood mononuclear cells and with its common polymorphisms. Analyses were performed in ethnically homogenous Polish Caucasians. The mean level of the WRN messenger RNA was significantly lower in long-living individuals than in young and middle-aged controls (p < .001 and p = .025, respectively). Analysis of the 361 bp WRN promoter CpG island showed that aging might be accompanied by a slight increase of its methylation status; however, it seems to be biologically insignificant. Finally, analysis of the WRN R834C, L1074F, and C1367R polymorphisms showed that the frequencies of the L1074F and C1367R polymorphisms were similar in all age groups tested, whereas the R834C polymorphism was absent from Polish Caucasians. We suggest that age-related decrease of the WRN expression but not its common genetic variants might contribute to human immunosenescence.

The -351A/G polymorphism of ESR1 is associated with risk of myocardial infarction but not with extreme longevity.

BACKGROUND: Women live longer than men. Some possible reasons for this advantage are the protection provided by high concentrations of 17ß-estradiol (E2) during the premenopausal period and polymorphic variants of the estrogen receptors (ERs), which mediate various cardiovascular functions of E2. METHODS: We tested whether the -351A/G and -397T/C polymorphisms of the ERα-encoding ESR1 were associated with extreme longevity. The genomic DNA of 148 centenarians (C), 414 young controls (Y), and 208 myocardial infarction patients (MI) was analyzed by RFLP-PCR. RESULTS: Both polymorphisms were equally distributed in the Y, C, and in centenarians never diagnosed with MI (HC). In centenarians, none of these polymorphisms was associated with a particular lipid profile. The AA genotype of the -351A/G polymorphism was less frequent in the C, HC and Y groups than in MI patients (p=0.058, p=0.021, and p=0.004, respectively). In MI patients, the GG genotype of the -351A/G polymorphism was associated with significantly lower mean total cholesterol, LDL, and HDL levels compared to the AG (p=0.0194, p=0.0213, and p=0.0367, respectively) and AA genotypes (p=0.0014, p=0.0078, and p=0.0448, respectively). CONCLUSIONS: The -351A/G ESR1 polymorphism might be associated with MI, but not with extreme longevity.

Decreased expression and the Lys751Gln polymorphism of the XPD gene are associated with extreme longevity.

Aging is associated with progressing genomic instability. The XPD gene encodes a DNA helicase involved in nucleotide excision repair and in transcription. We analyzed the common XPD polymorphisms that were previously shown to affect protein's DNA repair efficiency and to increase the risk of developing various cancers. Analysis was performed in 149 centenarians (mean age 101.1 years old) and in 413 young subjects (mean age 27.1 years old). We showed that the distribution of the Lys751Gln genotypes differed significantly between these groups (P = 0.017). In centenarians, the homozygous genotypes AA and CC were found less frequently than in young controls (29 vs. 36%, OR = 0.71, and 14 vs. 20%, OR = 0.652, respectively). The Arg156Arg and Asp312Asn were not significantly associated with extreme longevity. Analysis of the XPD mRNA level in blood mononuclear cells of people divided into three age groups (mean ages 28.7, 65.8 and 92.7 years old) showed that extreme longevity is associated with the decrease of the mean level of the specific mRNA; the differences between young or middle-aged vs. extremely old group were significant (P < 0.0001, P < 0.0001, respectively). In addition, the methylation pattern of the XPD promoter was analyzed in 30 people divided into three age groups (29.5, 65.9, and 101.4 years old). We showed that overall methylation of the XPD promoter is a rare event; however, aging is associated with the increase of methylation level upstream of the transcription start site. In summary, we showed for the first time that both the XPD polymorphic variants and the decreased level of its expression might be associated with aging.