bims-senagi Biomed News
on Senescence and aging
Issue of 2022‒05‒15
fifty-six papers selected by
Maria Grazia Vizioli
Mayo Clinic

  1. FASEB J. 2022 May;36 Suppl 1
      Cellular senescence is considered a hallmark of aging that often occurs in tissues and cells in response to DNA damage. Most healthy cells become senescent after a fixed number of divisions or irreparable genomic damage, often because of external conditions such as circulating factors that are released by adjacent senescent cells. These secreted factors, known as the senescence-associated secretory phenotype (SASP), are capable of in vivo reprogramming of otherwise healthy cells, ultimately inducing senescence. The involvement of cellular senescence in a variety of aging-related diseases has been thoroughly examined, however the role of cellular senescence and the SASP in aging muscle remains understudied. Therefore, the objective of this study was to examine the role of metabolites as potential components of the SASP through an easily reproducible model of senescence in skeletal muscle myoblasts. C2C12 myoblasts were treated with an antitumour antibiotic, bleomycin, to cause DNA damage-induced senescence, or with a vehicle control. Cells and associated media were collected 24 hours after treatment for metabolomic profiling using capillary-electrophoresis - mass spectrometry (CE-MS). Samples were compared to untreated myoblasts or fresh growth media and normalized to total cell count and protein content prior to analysis. Pathway analysis using the KEGG database revealed a substantial impact on amino acid metabolism within senescent cells, specifically phenylalanine and histidine metabolism. The media associated with senescent cells had a similar impact on amino acid metabolism, but also showed a significant impact on several polyunsaturated fatty acids, including arachidonic acid and docosahexaenoic acid. Notably, trimethylamine N-oxide (TMAO), a metabolite previously shown to promote endothelial senescence/dysfunction and neuroinflammation, was detected at significantly higher levels in the media surrounding senescent myoblasts relative to vehicle-treated controls (fold change = 2.878, p < 0.05). These data suggest that DNA damage-induced senescence significantly impacts the metabolome of skeletal muscle cells. Future work will aim to evaluate the targeted impact of candidate metabolites as potential senescence inducers.
  2. FASEB J. 2022 May;36 Suppl 1
      BACKGROUND: Cellular senescence, one of the hallmarks of aging, refers to a permanent cell cycle arrest and is accelerated during the aging process. A senolytic, a small molecule that eliminates senescent cells, can be a potential strategy to extend healthy lifespan and ameliorate age-associated diseases. Black ginseng (BG) prepared via nine-times repetitive steaming and drying process from fresh ginseng (Panax ginsengC.A. Meyer) has been reported to its physiological benefits against reactive oxygen species (ROS), inflammation, and oncogenesis which are common cues to induce aging. Therefore, the present study was aimed to investigate the effect of BG on cellular senescence.METHODS: Senescence-associated β-galactosidase (SA-β-gal) staining was performed on primary mouse embryonic fibroblasts (MEFs) treated 5 μg/mL BG extract for 7 days, and then senescence was induced by γ-ray irradiation (20 Gy). For animal studies, C57BL/6 male mice were divided into three groups: 9-week-old mice orally administered distilled water (Young), 21-month-old mice received administration of distilled water (Old), and aged mice supplemented 300 mg/kg BG extracts (Old + BG). After 4 weeks of daily injection, PCR array and immunoblotting were conducted to evaluate mRNA expression and protein levels of targets associated with cellular senescence and cell cycle regulation in metabolic organs, liver, skeletal muscle (SKM), and white adipose tissue (WAT).
    RESULTS: In in vitro study, BG treatment reduced the density of SA-β-gal-positive MEFs after γ-ray irradiation (Fig. 1). In aged mice study, BG supplementation remarkedly downregulated age-related hepatic genes, especially in complement component 1q B (C1qb) and C (C1qc), compared to the aged control group. We further examined the canonical Wnt signaling, augmented in a mouse model of accelerated aging, since the complement C1q family activates Wnt signaling by binding to Frizzled receptors. By assessing the related markers, BG supplementation significantly decreased the hepatic β-catenin expression level; it also slightly reduced the levels of glycogen synthase kinase 3β (GSK3β) and its phosphorylated form (p-GSK3β). Recent studies have revealed that Wnt signaling involves in mammalian aging by regulating cell fate and proliferation choice. Thus, we evaluated the expressions of p53 and p21, which involves in cell cycle regulation, and both levels decreased in liver samples from Old + BG group compared to the Old group (Fig. 2). In support of this notion, BG treatment decreased the hepatic mRNA expression levels of senescence-associated secretory phenotypes (SASPs) such as matrix metallopeptidase 12 (Mmp12) and chemokine ligand 2 (Cxcl2). Consistent with the results in the liver, other metabolic organs, SKM and WAT, showed significantly lower p53 expression in Old + BG group over the Old group.
    CONCLUSION: The current study indicates that BG could be a potential candidate of senolytics to reversely regulate cellular senescence, resulting from downregulated complement system affecting Wnt signaling pathways and p53/p21 pathway in metabolic organs.
  3. Sci Rep. 2022 May 11. 12(1): 7704
      Aging of sensory organs is associated with a decline in mitochondrial function and the accumulation of dysfunctional mitochondria. Impaired mitophagy blocks the turnover of dysfunctional mitochondria and leads to their accumulation. Urolithin A (UA) induces mitophagy in various mammalian cells. This study was aimed at investigating the effect of the mitophagy activator, UA, on premature senescent auditory cells. The levels of cellular senescence-associated p53 and p21 significantly increased in H2O2-induced senescent House Ear Institute-Organ of Corti 1 (HEI-OC1) cells and cochlear explants. However, the levels of mitophagy-related molecules significantly decreased. UA significantly decreased the expression of senescence-associated p53 and p21, and increased the expression of mitophagy-related proteins, in H2O2-induced senescent cells and cochlear explants. The percentage of β-galactosidase-stained senescent cells also reduced in H2O2-treated cells and cochlear explants upon UA pre-treatment. The formation of mitophagosomes and mitophagolysosomes was restored upon UA pre-treatment of H2O2-induced senescent cells. The knockdown of mitophagy-related genes (Parkin and Bnip3) resulted in annulment of UA-induced anti-senescent activity. UA significantly increased the ATP content, mitochondrial DNA (mtDNA) integrity, and mitochondrial membrane potential in senescent HEI-OC1 cells. These findings indicate that UA counteracted mitophagy decline and prevented premature senescence in auditory cells. Hence, UA administration might be a promising strategy for preventing mitochondrial dysfunction in patients with age-related hearing loss.
  4. Cells. 2022 Apr 30. pii: 1514. [Epub ahead of print]11(9):
      Immune checkpoint blockade (ICB) therapy is a central pillar of melanoma treatment leading to durable response rates. Important mechanisms of action of ICB therapy include disinhibition of CD4+ and CD8+ T cells. Stimulated CD4+ T helper 1 cells secrete the effector cytokines interferon-gamma (IFN-γ) and tumor necrosis factor alpha (TNF), which induce senescence in tumor cells. Besides being growth-arrested, senescent cells are metabolically active and secrete a large spectrum of factors, which are summarized as senescence-associated secretory phenotype (SASP). This secretome affects the tumor growth. Here, we compared the SASP of cytokine-induced senescent (CIS) cells with the SASP of therapy-induced senescent (TIS) cells. Therefore, we established in vitro models for CIS and TIS in melanoma. The human melanoma cell lines SK-MEL-28 and WM115 were treated with the cytokines IFN-γ and TNF as CIS, the chemotherapeutic agent doxorubicin, and the cell cycle inhibitor palbociclib as TIS. Then, we determined several senescence markers, i.e., growth arrest, p21 expression, and senescence-associated β-galactosidase (SA-β-gal) activity. For SASP analyses, we measured the regulation and secretion of several common SASP factors using qPCR arrays, protein arrays, and ELISA. Each treatment initiated a stable growth arrest, enhanced SA-β-gal activity, and-except palbociclib-increased the expression of p21. mRNA and protein analyses revealed that gene expression and secretion of SASP factors were severalfold stronger in CIS than in TIS. Finally, we showed that treatment with the conditioned media (CM) derived from cytokine- and palbociclib-treated cells induced senescence characteristics in melanoma cells. Thus, we conclude that senescence induction via cytokines may lead to self-sustaining senescence surveillance of melanoma.
    Keywords:  SASP; cell cycle inhibition; doxorubicin; immunotherapy; interferon; melanoma; palbociclib; senescence; tumor dormancy; tumor necrosis factor
  5. FASEB J. 2022 May;36 Suppl 1
      Advancing age leads to an accumulation of senescent endothelial cells (ECs) within arteries. Senescent cells have undergone permanent cell cycle arrest, are pro-oxidative and pro-inflammatory, and therefore represent a likely cause of age-related EC dysfunction. Yet, the molecular mechanisms and physiological consequences of EC senescence remain incompletely understood. Telomeres are repeat DNA sequences that cap chromosomes. Telomeres shorten with each cell division and are highly susceptible to oxidative damage. When telomeres become critically short or damaged, they become uncapped, which activates the DNA damage response and leads to cellular senescence. Here, we tested the hypothesis that aging results in EC telomere uncapping that induces senescence, leading to physiological hallmarks of aging. To assess whether aging results in EC telomere uncapping, we compared ECs from young (~3 mo) and old (~27 mo) mice. Aging resulted in ~4-fold greater EC telomere uncapping (p<0.001, Figure 1A). To determine if EC telomere uncapping induces senescence, we deleted the telomere capping protein, TRF2, in ECs of young (~3.5mo) mice (TRF2-ecKO). Compared to wildtype (WT) littermate controls, TRF2-ecKO mice had a ~78% reduction in TRF2 gene expression (p<0.0001). TRF2 deletion reduced EC division by ~47% (p<0.0001, Figure 1B), indicative of senescence. To examine the physiological consequences of EC senescence, we examined hallmarks of vascular aging including perfused microvascular density and endothelium-dependent dilation (EDD). TRF2-ecKO mice had an ~18% reduction in perfused mesenteric microvessels between 5-25 µM (p<0.001, Figure 1C). Likewise, TRF2-ecKO displayed ~26% reduction in mesenteric artery EDD compared to WT mice (p<0.05, Figure 1D). Furthermore, TRF2-ecKO arterial EDD was ameliorated by the superoxide scavenger, TEMPOL (p>0.05, Figure 1D). Endothelium-independent dilation to the exogenous nitric oxide donor sodium nitroprusside was not different between WT and TRF2-ecKO mice (p>0.05). These data suggest EC telomere uncapping leads to senescence that reduces perfused microvascular density, and to elevated oxidative stress that suppresses EDD, similar to advanced age. To assess metabolic and muscle function, we performed an intraperitoneal glucose tolerance test (GTT, 2g/kg body mass) and a forelimb grip strength test. TRF2-ecKO mice had ~21% greater area under the curve during GTT compared to WT mice (p<0.05, Figure 2A) as well as a ~14% reduction in grip strength (p<0.05, Figure2B). Taken together, these data provide evidence that aging results in EC telomere uncapping that induces senescence and age-related physiological dysfunction.
  6. Biochem Pharmacol. 2022 May 09. pii: S0006-2952(22)00172-1. [Epub ahead of print] 115078
      Endothelial cell senescence contributes to chronic inflammation and endothelial dysfunction, while favoring cardiovascular disorders and frailty. Senescent cells acquire a pro-inflammatory secretory phenotype that further propagates inflammation and senescence to neighboring cells. Cell senescence can be provoked by plethora of stressors, including inflammatory molecules and chemotherapeutic drugs. Doxorubicin (Doxo) is a powerful anthracycline anticancer drug whose clinical application is constrained by a dose-limiting cardiovascular toxicity. We here investigated whether cell senescence can contribute to the vascular damage elicited by Doxo. In human umbilical vein endothelial cells (HUVEC) cultures, Doxo (10-100 nM) increased the number of SA-β-gal positive cells and the levels of γH2AX, p21 and p53, used as markers of senescence. Moreover, we identified Doxo-induced senescence to be mediated by the nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasome, a key player of the immune innate system capable of releasing interleukin (IL)-1β. In fact, IL-1β itself mimicked the stimulatory action of Doxo on both NLRP3 activation and cellular senescence, while the pharmacological blockade of IL-1 receptors markedly attenuated the pro-senescence effects of Doxo. In search of additional pharmacological strategies to attenuate Doxo-induced endothelial senescence, we identified resolvin E1 (RvE1), an endogenous pro-resolving mediator, as capable of reducing cell senescence induced by both Doxo and IL-1β by interfering with the increased expression of pP65, NLRP3, and pro-IL-1β proteins and with the formation of active NLRP3 inflammasome complexes. Overall, RvE1 and the blockade of the NLRP3 inflammasome-IL-1β axis may offer a novel therapeutic approach against Doxo-induced cardiovascular toxicity and subsequent sequelae.
    Keywords:  Doxorubicin; Endothelial senescence; Interleukin 1β; NLRP3 inflammasome; Resolvin E1; Vascular aging
  7. FASEB J. 2022 May;36 Suppl 1
      Obesity is a major risk factor for the development of cardiovascular diseases like hypertension. It is estimated that obesity accounts for approximately 65-78% of hypertension in adults. Accumulating evidence suggests that increase in sympathetic nerve activity (SNA) plays a crucial role in the pathophysiology of obesity-induced hypertension. Previous studies from our lab have shown that obesity induces cellular senescence at the level of the rostral ventrolateral medulla (RVLM), which is responsible for basal and reflex control of sympathetic activity associated with cardiovascular function. Cellular senescence is a state of permanent, irreversible cell cycle arrest in proliferative cells. Senescent cells acquire senescence-associated secretory phenotype (SASP) and secrete proinflammatory cytokines, chemokines and proteases. These SASP factors could serve as a potential source of neuroinflammation. Based on the previous studies, our hypothesis is that obesity-induced senescence in the RVLM contributes to neuroinflammation and increases in SNA leading to the development of hypertension. To address this hypothesis, we eliminated senescent cells using a pharmacological approach (senolytic drug) and investigated its effect on sympathoexcitation and hypertension in obesity. Two-month-old male C57BL/6J mice were fed a high-fat diet (HFD; 60% fat) or a chow diet (Controls; 10% fat) for 4-6 months. After 4-6 months of feeding, the two groups were again subdivided into two subgroups: Vehicle and a senolytic drug combination of Dasatinib+Quercetin (D+Q). So, the four different groups are Control + Vehicle, Controls + (D+Q), HFD + Vehicle, and HFD + (D+Q). Dasatinib (5 mg/kg) plus Quercetin (50 mg/kg) was prepared in a diluted solution comprising 10% ethanol, 30% polyethylene glycol 400, and 60% Phosal 50 PG. The intervention (Vehicle or D+Q) was performed for 3 consecutive days and for 2 cycles with a 2-week interval between cycles. Hemodynamic parameters (Blood pressure & Heart rate) and SNA were analyzed by radiotelemetry. As expected, the low frequency/high frequency heart rate variability (LF/HF HRV) ratio which is an indicator of SNA was significantly higher in HFD animals, which was reversed by senolytic drug treatment with D+Q. No changes were observed in the blood pressure. These results suggest that obesity-induced cellular senescence causes sympathoexcitation which was reversed by the removal of senescent cells using senolytics. Future studies will investigate the mechanistic role of cellular senescence at the level of RVLM in obesity-induced sympathoexcitation.
  8. FASEB J. 2022 May;36 Suppl 1
      OBJECTIVES: Cellular senescence is a stress response involving permanent replicative arrest. It is accompanied by a complex senescence-associated secretory phenotype (SASP), which is characterized by the presence of pro-inflammatory cytokines and chemokines, growth factors, and tissue-remodeling metalloproteinases. Little is known regarding the role of senescence and the SASP in the context of arrhythmias post myocardial infarction (MI). Here, we characterized the arrhythmogenic cardiac consequences and molecular mechanisms underlying tissue remodeling changes in impaired or accelerated cardiac myofibroblast (CMF) senescence and the pro-inflammatory SASP during post-MI wound healing process in genetically engineered mice.MATERIAL-METHODS: Impaired or accelerated CMF-specific senescence was achieved by using cre-lox mouse models to delete p53 or MDM2 genes. MI surgery via direct ligation of the left anterior descending artery (LAD) was performed in 4-month-old wild-type (WT), Pstn-Cre(+/-) p53(fl/fl), Pstn-MerCreMer(+/-) MDM2(fl/fl), and respective littermate control (LMC) mice of both sexes. Changes in cardiac structure and function post-MI were assessed by echocardiography. Senescence-associated β-galactosidase (SA-βgal) staining was applied to detect and quantify senescent cells in three zones: infarct zone (IZ), infarct border zone (IBZ), and remote zone (RZ). Immunofluorescence staining identified the types of senescent cells. Masson's trichrome staining was used for fibrosis assessment. RT-qPCR was performed to quantitate the expression of known senescence genes and canonical SASP factors.
    RESULTS: Pstn-Cre(+/-) p53(fl/fl) mice displayed significantly decreased CMF senescence two weeks post-MI in the scar compared to littermate control p53(fl/fl) and WT mice. We noticed a similar trend in the IBZ, but this did not reach statistical significance. Pstn-MCM(+/-) MDM2(fl/fl) mice showed significantly increased CMF senescence in the IZ compared littermate control MDM2(fl/fl) mice. We observed a similar trend in the IBZ, but this did not reach statistical significance. In all mice, we observed essentially no senescence in the RZ. Our data did not show any significant difference in fibrosis between any genotype in any zone. We did not observe any overall trend in differential expression in senescence and SASP genes between p53(fl/fl) and Pstn-Cre(+/-) p53(fl/fl) mice in any zone. There was no evidence of any sex-specific differences in aforementioned results.
    CONCLUSIONS: A significant increase and persistence of CMF senescence likely contributes to pathological tissue remodeling during the post-MI wound healing process. This is likely due to SASP-induced pro-arrhythmogenic chronic inflammation or via direct cell-cell interactions between senescent CMFs and myocytes.
  9. FASEB J. 2022 May;36 Suppl 1
      Cellular senescence, induced by stress factors within a cell, is a state of cell growth arrest that has been found to contribute to age-related diseases. Senescent cells exhibit a particular phenotype that is generically characterized by increased expression of cell-cycle regulator proteins, increased expression of senescence associated beta-galactosidase, and increased oxidative mitochondrial stress. Particularly in the cardiomyocyte, this state of growth arrest contributes to many prevalent cardiomyopathies. However, while it is broadly known that oxidative stress and DNA damage inducing conditions can contribute to a cell becoming senescent, methods to induce senescence in cardiomyocytes effectively have yet to be systematically investigated. In particular, the particular lesions and concentrations that can adequately induce senescence as well as the particular pathway through which senescence is induced in each induction method are unknown. Accordingly, the aim of this study is to optimizethe dosage and regimen of three pro-senescence insults (hydrogen peroxide, doxorubicin, and UV-C light) that will best induce senescence as measured by the associated senescent phenotype while gaining deeper understanding of differences in the pathways that are employed within a cell in the process of inducing senescence from different forms of stress. To do so, we used western blots and staining techniques in order to analyze relative expression of classic senescence markers in cells that were exposed to stress conditions as compared to healthy cells. The most relevant proteins that were compared were p16 and p21, cell cycle regulator proteins, and H2A.X, a double-stranded DNA damage protein. We found that in conditions of 500 μM H2O2, 0.1 μM Dox, and 5 mJ/cm^2 UV-C light, p21 and H2A.X were significantly upregulated and p16 was interestingly found to be significantly downregulated in all conditions. Treated samples were then filtered and tested via mass spectrometry in order to determine relative protein expressions. Preliminary mass spectrometry data used to compare the different insults showed some evidence of activation of different senescent pathways. Samples insulted with Dox showed an enrichment in the apoptotic pathway while those treated with UV-C or H2O2 showed enrichment in translation elongation and nonsense mediated decay related pathways. Furthermore, we found that combinatorial treatment of H2O2, UV-C light and doxorubicin increased expressed mitochondrial oxidative stress as seen by MitoSox staining techniques. In summary, our results showed the different concentrations that can induce senescent phenotypes in cardiomyocytes and the differences in the phenotypes through different senescence induction methods.
  10. FASEB J. 2022 May;36 Suppl 1
      Doxorubicin (DOX) is a highly effective chemotherapeutic agent; however its clinical use is limited due to cardiorespiratory complications, including the development of dyspnea, exercise intolerance, and cardiomyopathy. While the mechanisms responsible for DOX-induced cardiorespiratory dysfunction remain unclear, evidence indicates that DOX administration causes DNA damage and promotes oxidative stress-induced cellular senescence. Factors shown to contribute to DOX-induced cellular senescence include cell cycle arrest, driven by signaling through the p16INK4a/Rb and p21/p53 pathways, impaired mitochondrial function, upregulation of pro-survival pathways, increased lysosomal mass, and expression of the proinflammatory senescence-associated secretory phenotype (SASP). In contrast, exercise has been shown to prevent cellular senescence in several tissues, leading to the hypothesis that endurance exercise preconditioning is sufficient to prevent senescence induced in the heart and diaphragm muscle following DOX chemotherapy treatment. To determine the relationship between DOX-induced cardiorespiratory impairment, exercise preconditioning and the impact of accelerated cellular senescence, female Sprague-Dawley rats underwent two weeks of endurance exercise training (treadmill running, 5 days/week, 60 min/day, 30m/min, 0% grade) before receiving a bolus dose of DOX (20mg/kg IP) or saline treatment. Functional measures were evaluated 48 hours after DOX or saline administration, followed by tissue collection for subsequent analysis of cellular senescence markers. Our results revealed that exercise preconditioning prior to DOX administration prevented diastolic and systolic dysfunction in the heart, with marked improvements in fractional shortening, myocardial performance index, and posterior wall shortening velocity compared to sedentary DOX-treated animals. DOX-induced weight loss, reduction in grip strength, and diaphragm weakness were partially rescued with exercise as well, with diaphragm specific force production preserved at frequencies of 60-160Hz. Finally, both exercise and DOX appear to alter expression of proteins associated with the senescent phenotype in the heart and diaphragm. Therefore, the protective effects of exercise against DOX cardiorespiratory dysfunction may be related to changes in muscle cellular senescence.
  11. FASEB J. 2022 May;36 Suppl 1
      NAD+ is an essential coenzyme found in all living cells. NAD+ concentrations decline during aging, but whether this reflects impaired production or accelerated consumption remains unclear. Here we employed isotope tracing and mass spectrometry to probe NAD+ metabolism across tissues in aged mice. In 25-month-old mice, we observe modest tissue NAD+ depletion (median decrease ~30%) without significant changes in circulating NAD+ precursors. Isotope tracing showed unimpaired synthesis of circulating nicotinamide from tryptophan, and maintained flux of circulating nicotinamide into tissue NAD+ pools. Although absolute NAD+ biosynthetic flux was maintained in most tissues of aged mice, fractional tissue NAD+ labeling from infused labeled nicotinamide was modestly accelerated, consistent with increased activity of NAD+ consuming enzymes. Long-term calorie restriction partially mitigated age-associated NAD+ decline despite decreasing NAD+ synthesis, suggesting that calorie restriction reduces NAD+ consumption. Acute inflammatory stress induced by LPS decreased NAD+ by impairing synthesis in both young and aged mice. Thus, age-related decline in NAD+ is relatively subtle and driven by increased NAD+ consumer activity rather than impaired production.
  12. FASEB J. 2022 May;36 Suppl 1
      Aging is a physiological decrease of several biological activities in an organ that all organisms go through. The presence of a gradual deterioration of cell functioning, due to damage accumulation in metabolic organs, accelerates biological aging. Recently, dietary interventions with food-active compounds have been linked to suppressing the accumulation of senescent cells and senescence-associated secretory phenotype (SASP). Curcumin has potent biochemical and biological activities, including antioxidant and anti-inflammatory actions. However, it largely remains unclear how curcumin has anti-aging properties such as protection of DNA damage and cell survival/cell fate decisions. The objective of this study is to examine the regulatory effect of dietary curcumin on hepatic cellular senescence in the aged mouse model. Aged (18-20 months old) male C57BL/6 mice were fed a normal chow diet (NCD) or NCD containing 0.4% (w/w) curcumin - equivalent to 2g/day for a 60 kg adult - (NCD+CUR), high fat high sugar diet (HFHSD) or a HFHSD+CUR (N=7-9 per group) for 15 weeks. Mice given an HFHSD supplemented with curcumin displayed a different metabolic phenotype compared to mice given an HFHSD alone. To examine the phenotypic plasticity led by transcriptomic alteration, we used RNA-Seq and analyzed differential gene expression in Gene Ontology (GO) terms and KEGG pathway analysis. There were 1687 and 3794 number of genes that showed a significant change with curcumin in NCD and HFHSD groups compared to their respective control groups. There were at least 8-fold higher uniquely upregulated and 6-fold uniquely downregulated genes in the HFHSD+CUR group when compared with their NCD counterparts. To this extent, curcumin supplementation altered hepatic gene expression profiling, especially in senescence pathways and their associated genes. Thus, to validate downregulation in senescence pathway involved genes from the RNA-Seq data results, we compared the distribution of hepatic senescent cells by β-gal staining in HFHSD groups. It revealed that HFHSD+CUR mice showed less density of the hepatic senescent cell (Figure A). We then mechanistically sought how curcumin regulates the hepatic senescence pathway. We found that curcumin supplementation decreased senescence effectors, specifically p38 and JNK protein expression levels in the liver (Figure B, C). Our findings suggest that the multifaceted therapeutic potential of curcumin can be used as a protective agent for age-induced metabolic diseases.
  13. Aging Cell. 2022 May 08. e13626
      The proportion of humans suffering from age-related diseases is increasing around the world, and creative solutions are needed to promote healthy longevity. Recent work has clearly shown that a calorie is not just a calorie-and that low protein diets are associated with reduced mortality in humans and promote metabolic health and extended lifespan in rodents. Many of the benefits of protein restriction on metabolism and aging are the result of decreased consumption of the three branched-chain amino acids (BCAAs), leucine, isoleucine, and valine. Here, we discuss the emerging evidence that BCAAs are critical modulators of healthy metabolism and longevity in rodents and humans, as well as the physiological and molecular mechanisms that may drive the benefits of BCAA restriction. Our results illustrate that protein quality-the specific composition of dietary protein-may be a previously unappreciated driver of metabolic dysfunction and that reducing dietary BCAAs may be a promising new approach to delay and prevent diseases of aging.
    Keywords:  FGF21; amino acids; branched-chain amino acids; isoleucine; lifespan; mTOR
  14. FASEB J. 2022 May;36 Suppl 1
      BACKGROUND: Hepatic encephalopathy resultant from chronic liver failure (Type C HE) is associated with neurological dysfunction and hyperammonemia. In HE, senescent markers have been found in astrocytes, but research into neuronal senescence has been limited. TGFb1 is one of the senescence-associated secretory phenotype (SASP) factors released from senescent cells, and has been shown to be upregulated in the brain in Type A HE, but its role in Type C HE is unknown. The aim of this study was to assess the role of a senescence-like phenotype in the pathogenesis of Type C HE.METHODS: SD rats underwent bile duct ligation (BDL) surgery and C57BL/6 mice were treated with CCl4 for 16 wks to induce chronic liver failure. CCl4 -treated mice received a senolytic cocktail of Dasatinib and qercertin (D/Q via oral gavage) starting after 8 weeks of CCl4 injections. In parallel, human autopsy brain samples from patients with HE due to liver cirrhosis and age- and sex-matched controls were used. Neurobehavioral testing to assess cognitive and neuromuscular function was performed. Markers of senescence (p21, p16, b-galactosidase expression), SASPs (CCL2, TGFb1, and IL-6) and neuroinflammation were assayed in the cerebellum and hippocampus by qPCR and immunohistochemistry.
    RESULTS: In rodent models of HE and in human autopsy samples, markers of senescence and SASP were observed in neurons of the CA1 and dentate gyrus in the hippocampus and in the purkinje cell layer in the cerebellum, regions known for their involvement in learning and memory and motor control. Treatment with D/Q reduced the number of senescent neurons, and attenuated the cognitive deficits, the neuromuscular impairment, presence of ataxia and neuroinflammation observed in the mouse model of HE.
    CONCLUSION: Taken together, these data suggest that the acquisition of a senescent-like phenotype in neurons in the hippocampus and cerebellum in models of Type C HE may contribute to the pathogenesis of Type C HE. Taken together our data suggests that targeting the senescence-like phenotype in HE may be a viable option for the development of novel therapeutic strategies.
  15. Cells. 2022 Apr 30. pii: 1505. [Epub ahead of print]11(9):
      Human ageing can be characterized by a profile of circulating microRNAs (miRNAs), which are potentially predictors of biological age. They can be used as a biomarker of risk for age-related inflammatory outcomes, and senescent endothelial cells (ECs) have emerged as a possible source of circulating miRNAs. In this paper, a panel of four circulating miRNAs including miR-146a-5p, miR-126-3p, miR-21-5p, and miR-181a-5p, involved in several pathways related to inflammation, and ECs senescence that seem to be characteristic of the healthy ageing phenotype. The circulating levels of these miRNAs were determined in 78 healthy subjects aged between 22 to 111 years. Contextually, extracellular miR-146a-5p, miR-126-3p, miR-21-5p, and miR-181a-5p levels were measured in human ECs in vitro model, undergoing senescence. We found that the levels of the four miRNAs, using ex vivo and in vitro models, progressively increase with age, apart from ultra-centenarians that showed levels comparable to those measured in young individuals. Our results contribute to the development of knowledge regarding the identification of miRNAs as biomarkers of successful and unsuccessful ageing. Indeed, they might have diagnostic/prognostic relevance for age-related diseases.
    Keywords:  ageing; endothelial senescence; inflamm-ageing; longevity; miRNAs
  16. FASEB J. 2022 May;36 Suppl 1
      It is estimated that more than 50% of adults over 80 will develop dementia, including Alzheimer's disease (AD), and the primary risk factor for dementia is aging. Aging provides the substrate for disease through increased inflammation and cellular dysfunction, which in turn sets the stage for neurodegenerative disease. In a previous study we validated replicative senescence, a model of cell senescence that permits the study of cells as they progress to senescence, in human coronary artery endothelial cells (HCAEC). Here, we tested the hypothesis that senescent endothelial cells, which accumulate with aging and release senescence-associated secretory phenotype (SASP) and extracellular vesicles, create a pro-inflammatory environment that adversely affects the viability and morphology of neurons, which are endpoints implicated in AD. We categorized the phenotypes of HCAEC by passage number and identified them as EP- early passage (non-senescent), ES- early senescence and LS- late senescence. HCAEC at these varying stages of senescence were co-cultured with LUHMES cells, a human neuronal cell line. Co-culturing HCAEC LS with LUHMES significantly decreased LUHMES viability, while co-culturing HCAEC ES with LUHMES decreased the number of neurites extended by LUHMES in 24h and decreased the length of LUHMES' neurites in 48h, when compared to LUHMES grown in the absence of HCAEC for the same period of time. HCAEC EP or LS co-cultured with LUHMES did not affect the number or length of the LUHMES' neurites, when compared to LUHMES grown in the absence of HCAEC. Using a cytokine array containing probes for 105 analytes, we detected 17 inflammatory mediators released by the co-cultures of HCAEC (EP, ES and LS) with LUHMES. Of these 17 mediators, 14 were also found at supernatants of HCAEC (EP, ES and LS) grown in the absence of LUHMES, and none of the analytes were detected in supernatants from LUHMES grown in the absence of HCAEC. Statistical analysis of the intensity of the positive reaction for inflammatory mediators in the array was run for all the 17 positive analytes, and only IL-8 and Serpin E1 release into the supernatants of the co-cultures were significantly increased in co-cultures of LUHMES and senescent HCAEC (ES or LS), when compared to the levels of the same mediators released by co-cultures of LUHMES and EP HCAEC. However, exposing LUHMES to IL-8 and Serpin E1, in the absence of HCAEC, did not affect LUHMES viability, the number or length of LUHMES' neurites or LUHMES' NF-kB activation. The results from the LUHMES and HCAEC co-cultures study supports a role for aging endothelial cells in the toxicity and morphological remodeling of the surrounding neurons, which may contribute to the development and progression of neurodegenerative diseases. However, our data exclude IL-8 and Serpin E1 as direct independent mediators of senescent HCAEC on neuronal cytotoxicity and neurite remodeling.
  17. FASEB J. 2022 May;36 Suppl 1
      INTRODUCTION: Despite the advent of stents, intimal hyperplasia subsequent to vascular interventional procedures remains a major obstacle to success. Vascular smooth muscle cells (VSMC) play a critical role in the development and pathogenesis of intimal hyperplasia indicative of restenosis; therefore regulation of gene expression in VSMC represents a logical intervention point to attenuate this syndrome. FXR1 is a muscle-enhanced RNA binding protein and expression is increased in injured human and mouse arteries. We have shown that modulation of FXR1 levels affects abundance and stability of inflammatory transcripts in VSMC, suggesting that FXR1 is a negative regulator of inflammation. This drives our hypothesis that FXR1 is involved in mitigating vascular disease and acts as a crucial regulator of inflammatory and proliferative mRNA in VSMC.APPROACH/RESULTS: Because FXR1 expression is increased in injured arteries, we have developed a novel VSMC-specific conditional knockout mouse (FXR1VSMC/VSMC ). In a carotid artery ligation model of intimal hyperplasia, FXR1VSMC/VSMC mice have significantly reduced neointima formation (p<0.001) post-ligation compared to several controls. To determine the mechanism of these effects, we knocked down FXR1 in human VSMC by siRNA and observed decreased proliferation (p<0.05) as well as an increase in beta galactosidase (p<0.05) and gamma H2AX (p<0.01) staining compared with controls, indicative of senescence. Senescent cells exhibit phenotypic changes called the senescence associated secretory phenotype (SASP) with characteristic gene expression leading to increased inflammation in the tissue microenvironment. RIP-sequencing demonstrated that FXR1 interacts with transcripts involved in cell cycle control, and RNA stability of these transcripts is decreased with FXR1 KO. qPCR analysis from FXR1 KO mouse VSMC show an increase in transcripts associated with senescence (p21, p16, p53) as well as an increase in SASP-associated mRNA compared with controls. Furthermore, wild-type human VSMC cultured in conditioned media from cells transfected with FXR1 siRNA show an increase in SASP mRNA and an increase in proliferation compared with cells cultured in conditioned media from cells transfected with a scrambled control.
    SUMMARY & CONCLUSIONS: Our results are the first to suggest that in addition to destabilization of inflammatory transcripts, FXR1 may stabilize cell cycle related genes in VSMC, and absence of FXR1 leads to induction of a senescent phenotype, an increase in SASP gene expression, and reduction of intimal hyperplasia.
  18. FASEB J. 2022 May;36 Suppl 1
      Age-related vascular dysfunction (e.g., large elastic artery [aorta] stiffening and endothelial dysfunction) is mediated by excess reactive oxygen species (ROS) leading to lower nitric oxide (NO) bioavailability. The upstream mechanisms mediating excess ROS are mostly unknown. Cellular senescence is a principal mechanism of aging and the senescence associated secretory phenotype (SASP) may exacerbate ROS.PURPOSE: To: 1) determine if senescent cell clearance (senolysis) lowers aortic stiffness (pulse wave velocity [PWV]) and increases endothelial function (endothelium-dependent dilation [EDD]), and if these effects are mediated by reduced ROS and increased NO bioavailability; and 2) isolate the influence of the circulating SASP on age-related vascular dysfunction.
    METHODS AND RESULTS: Young (6 mo) and old (27 mo) adult male and female p16-3MR mice were treated with vehicle ([V]; saline) or a p16-3MR senolytic, ganciclovir (GCV; 25 mg/kg/day) injected intraperitoneally for 5 days. This resulted in 4 groups/sex (Young-V [YV], n = 23; Young-GCV [Y-GCV]; n = 16, Old-V [OV]; n = 22, Old-GCV [O-GCV]; n = 21). No sex differences were observed, so results were combined. Aortic Stiffness. Aortic PWV (aPWV) was assessed pre and post V and GCV treatment. Old mice had higher aPWV at baseline vs young (aPWV [cm/sec]: O-GCV, 450 ± 16; OV, 441 ± 13; YV, 352 ± 7; Y-GCV, 351 ± 12; P<.05). Following GCV treatment, old mice had reduced aPWV (pre: 441 ± 13 vs post: 375 ± 5 cm/sec, P< .05), which was not different from YV (P= .63) or Y-GCV (P= .72). These data suggest that cellular senescence mediates aortic stiffening with advancing age. Endothelial function. OV animals had impaired ex vivocarotid artery EDD to acetylcholine (an established assay of endothelial function) relative to young and GCV treatment resulted in greater peak EDD (Peak EDD [%]: O-GCV, 95 ± 1; OV, 83 ± 4; YV, 93 ± 4; Y-GCV, 95 ± 1; P<.05). Addition of the NO-synthase inhibitor, L-NAME, abolished group differences suggesting the age-related increase in cellular senescence reduced EDD by lowering NO bioavailability. Administration of the ROS scavenger TEMPOL eliminated group differences in EDD, implying that cellular senescence causes endothelial dysfunction with aging by amplifying ROS. SASP. To determine the effect of the circulating SASP on aortic stiffness, we incubated aortic rings from young adult (6 mo) male and female C57BL/6 mice, with plasma from sex-matched OV and O-GCV mice, or under fetal bovine serum (control). Following a 48h incubation, we assessed intrinsic mechanical wall stiffness (elastic modulus [EM]), an established ex vivomeasure of aortic stiffness. OV plasma resulted in 1.5 ± .01-fold greater EM vs control (P< .05), which did not occur in O-GCV plasma (P= 0.84 vs control), suggesting the SASP may increase age-related aortic stiffening.
    CONCLUSION: . The circulating SASP factors may be a mechanism by which cellular senescence induces age-related vascular dysfunction, and as such, these processes represent novel therapeutic targets.
  19. FASEB J. 2022 May;36 Suppl 1
      Protracted quadriceps weakness and atrophy after anterior cruciate ligament (ACL) injury result in poor knee mechanics and contribute to the development of posttraumatic osteoarthritis. The biological underpinnings of poor functional recovery and osteoarthritis are unknown, limiting the development of evidence-based therapies. The identification of shared cellular pathology between skeletal muscle, bone and articular cartilage would offer powerful therapeutic targets to combat musculoskeletal degeneration; therefore, we sought to define the musculoskeletal cellular senescence burden as a cellular predictor of muscle atrophy and osteoarthritis severity. We hypothesized that cellular senescence would be associated with greater muscle atrophy and osteoarthritis severity. Male and female C57BL/6 mice between 4 and 6 months of age underwent unilateral ACL transection surgery (ACLT), and then quadriceps and whole knees (healthy contralateral and ACLT) were collected at 7, 14 and 28d post-ACLT. Quadriceps fiber size was determined using immunohistochemistry (IHC), and osteoarthritis severity was assessed with Safranin O staining via Osteoarthritis Research Society International (OARSI) scoring. Additionally, cellular senescence was determined by quantification of senescence-associated β-galactosidase (SA β-Gal) and p16Ink4a (p16) positive cells using IHC. SA β-Gal expression was greatest at 7 days post-ACLT (ACLT: 18.6±4.3 vs. Healthy: 3.3±2.6 cells/mm2 , p<0.05) but remained elevated through 28 days post-ACLT. Quadriceps senescent cell density occurred alongside atrophy (7 days: -21%, 14 days: -16%, 28 days: -13%, all p<0.05 vs healthy contralateral). p16+ cell density was elevated within the articular cartilage (ACLT: 32.0±5.8 vs. Healthy: 10.0±2.5 cells/mm2 , p<0.05) and trabecular bone (ACLT: 435.5±130.6 vs. Healthy: 119.4±8.6 cells/mm2 , p=0.08) at 28 days post-ACLT. OARSI scoring demonstrated cartilage erosion at 28 days post-ACLT (ACLT: 2.9±1.0 vs Healthy: 0.4±0.4 OARSI score, p<0.05). These findings support an elevated cellular senescence burden within muscle, bone and cartilage following ACL injury associated with musculoskeletal deficits and osteoarthritis. Future research should focus on strategies to target senescent cells to enhance functional recovery and mitigate osteoarthritis following ACL injury.
  20. FASEB J. 2022 May;36 Suppl 1
      Sex differences in the development of hypertension across the lifespan are well established. Women are at lower risk of hypertension compared to age-matched men until menopause, when the risk of hypertension sharply rises for women. The peptide hormone angiotensin II (Ang II) is a well-recognized contributor to hypertension, particularly through its actions in the brain. Importantly, Ang II is too large to cross into the central nervous system (CNS) and influences blood pressure regulation by acting at circumventricular nuclei located outside of the blood-brain-barrier, particularly the forebrain subfornical organ. For instance, in males, Ang II drives pro-hypertensive cellular stressors in the SFO while females are resistant to Ang II-induced hypertension. However, the underlying CNS (i.e. SFO) mechanisms that contribute to hypertension in a sexually dimorphic manner remain unclear. Intriguingly, Ang II-induced stressors can culminate in cellular senescence. Cellular senescence is a complex cellular phenotype characterized by marked changes in cell metabolism, macromolecular damage, and a pro-inflammatory environment known as the senescence associated secretory phenotype (SASP). The role of senescence in the CNS as related to the development of hypertension remains unclear. Based on this, we hypothesized that brain cellular senescence, particularly in the SFO, may be a novel mechanism for the sexually dimorphic nature of Ang II-induced hypertension. To test this, 8-week-old male and female C57Bl/6J mice (n=7-10/group) were implanted with subcutaneous mini-osmotic pumps for the infusion of Ang II (600 ng/kg/min). Micropunches of the SFO were collected at baseline and following 14 days of Ang II infusion for quantitative real-time PCR analysis. In males, infusion of Ang II resulted in a robust increase in key senescent genes including p16 (1.9±0.3 fold baseline, p<0.05) and p21 (2.5±0.5 fold baseline, p<0.05) in the SFO. In parallel with the induction of senescence, inflammatory SASP indicators were also upregulated in the SFO of males following 14 days of Ang II infusion, including interleukin-6 (IL-6: 2.4±0.4 fold baseline, p<0.05) and interleukin-10 (IL-10: 2.8±0.7 fold baseline, p<0.05). However, when examining females, clear sexual dimorphism in Ang II-induced SFO cellular senescence was apparent. Specifically, no changes in p16 (1.1±0.4 fold baseline, p=0.9) or p21(1.2±0.2 fold baseline, p=0.2) were noted in the SFO following Ang II administration. Similarly, SASP markers that accompany senescence remained unchanged in the SFO (e.g. IL-6: 1.3±0.5 fold baseline, p=0.8 and IL-10: 0.98±0.2 fold baseline, p=0.6). Together, these findings indicate that: 1) Cellular senescence in the SFO is associated with Ang II-induced hypertension in males; and 2) Females are protected against Ang II-driven cellular senescence in the SFO. Importantly, cellular senescence increases with advancing age. Thus, our data suggests that cellular senescence may underlie the sexually dimorphic nature of hypertension at younger ages, as well as the increased hypertensive risk in women as they age.
  21. Aging Cell. 2022 May 13. e13624
      One of the earliest hallmarks of immune aging is thymus involution, which not only reduces the number of newly generated and exported T cells, but also alters the composition and organization of the thymus microenvironment. Thymic T-cell export continues into adulthood, yet the impact of thymus involution on the quality of newly generated T-cell clones is not well established. Notably, the number and proportion of medullary thymic epithelial cells (mTECs) and expression of tissue-restricted antigens (TRAs) decline with age, suggesting the involuting thymus may not promote efficient central tolerance. Here, we demonstrate that the middle-aged thymic environment does not support rapid motility of medullary thymocytes, potentially diminishing their ability to scan antigen presenting cells (APCs) that display the diverse self-antigens that induce central tolerance. Consistent with this possibility, thymic slice assays reveal that the middle-aged thymic environment does not support efficient negative selection or regulatory T-cell (Treg) induction of thymocytes responsive to either TRAs or ubiquitous self-antigens. This decline in central tolerance is not universal, but instead impacts lower-avidity self-antigens that are either less abundant or bind to TCRs with moderate affinities. Additionally, the decline in thymic tolerance by middle age is accompanied by both a reduction in mTECs and hematopoietic APC subsets that cooperate to drive central tolerance. Thus, age-associated changes in the thymic environment result in impaired central tolerance against moderate-avidity self-antigens, potentially resulting in export of increasingly autoreactive naive T cells, with a deficit of Treg counterparts by middle age.
    Keywords:  T cell; cellular immunology; central tolerance; immune aging; thymus involution
  22. Geroscience. 2022 May 11.
      Aging is a progressive loss of physiological function and increased susceptibility to major pathologies. Degenerative diseases in both brain and bone including Alzheimer disease (AD) and osteoporosis are common in aging groups. TERC is RNA component of telomerase, and its deficiency accelerates aging-related phenotypes including impaired life span, organ failure, bone loss, and brain dysfunction. In this study, we investigated the traits of bone marrow-brain cross-tissue communications in young mice, natural aging mice, and premature aging (TERC deficient, TERC-KO) mice by single-cell transcriptome sequencing. Differentially expressed gene analysis of brain as well as bone marrow between premature aging mouse and young mouse demonstrated aging-related inflammatory response and suppression of neuron development. Further analysis of senescence-associated secretory phenotype (SASP) landscape indicated that TERC-KO perturbation was enriched in oligodendrocyte progenitor cells (OPCs) and hematopoietic stem and progenitor cells (HSPC). Series of inflammatory associated myeloid cells was activated in premature aging mice brain and bone marrow. Cross-tissue comparison of TERC-KO mice brain and bone marrow illustrated obvious ligand-receptor communications between brain glia cells, macrophages, and bone marrow myeloid cells in premature aging-induced inflammation. Enrichment of co-regulation modules between brain and bone marrow identified premature aging response genes such as Dusp1 and Ifitm3. Our study provides a rich resource for understanding premature aging-associated perturbation in brain and bone marrow and supporting myeloid cells and endothelial cells as promising therapy targeting for age-related brain-bone diseases.
    Keywords:  Aging; Brain-bone axis; Single-cell RNA sequencing; TERC; Telomerase
  23. Aging (Albany NY). 2022 May 09. 14(undefined):
      A thought-provoking article by Gems and de Magalhães suggests that canonic hallmarks of aging are superficial imitations of hallmarks of cancer. I took their work a step further and proposed hallmarks of aging based on a hierarchical principle and the hyperfunction theory.To do this, I first reexamine the hallmarks of cancer proposed by Hanahan and Weinberg in 2000. Although six hallmarks of cancer are genuine, they are not hierarchically arranged, i.e., molecular, intra-cellular, cellular, tissue, organismal and extra-organismal. (For example, invasion and angiogenesis are manifestations of molecular alterations on the tissue level; metastasis on the organismal level, whereas cell immortality is observed outside the host).The same hierarchical approach is applicable to aging. Unlike cancer, however, aging is not a molecular disease. The lowest level of its origin is normal intracellular signaling pathways such as mTOR that drive developmental growth and, later in life, become hyperfunctional, causing age-related diseases, whose sum is aging. The key hallmark of organismal aging, from worms to humans, are age-related diseases. In addition, hallmarks of aging can be arranged as a timeline, wherein initial hyperfunction is followed by dysfunction, organ damage and functional decline.
    Keywords:  carcinogenesis; geroscience; hyperfunction theory; mTOR; oncology; rapamycin
  24. Aging Cell. 2022 May 10. e13616
      Accumulation of oxidative stress is highly intertwined with aging process and contributes to aging-related diseases, such as neurodegenerative diseases. Deciphering the molecular machinery that regulates oxidative stress is fundamental to further uncovering the pathogenesis of these diseases. Chaperone-mediated autophagy (CMA), a highly selective lysosome-dependent degradation process, has been proven to be an important maintainer of cellular homeostasis through multiple mechanisms, one of which is the attenuation of oxidative stress. However, the specific mechanisms underlying this antioxidative action of CMA are not fully understood. In this study, we found that CMA directly degrades Kelch-like ECH-associated protein 1 (Keap1), an adaptor of E3 ligase complex that promotes the degradation of nuclear factor erythroid 2-related factor 2 (Nrf2), which is a master transcriptional regulator in antioxidative response. Activated CMA induced by prolonged oxidative stress led to an increase in Nrf2 level by effectively degrading Keap1, contributing to Nrf2 nuclear translocation and the expression of multiple downstream antioxidative genes. Meanwhile, together with previous study showing that Nrf2 can also transcriptionally regulate LAMP2A, the rate-limiting factor of CMA process, we reveal a feed-forward loop between CMA and Nrf2. Our study identifies CMA as a previously unrecognized regulator of Keap1-Nrf2 pathway and reinforces the antioxidative role of CMA.
    Keywords:  6-OHDA; CMA; Keap1-Nrf2 pathway; oxidative stress
  25. FASEB J. 2022 May;36 Suppl 1
      Age-related macular degeneration (AMD) is the leading cause of blindness in industrialized countries. One of the potential contributors to AMD pathology is retinal pigment epithelial (RPE) cell senescence. Through experimental trial, it is shown that sulindac, an FDA approved non-steroidal anti-inflammatory drug, protects normal cells such as retinal, cardiac, and neuronal cells against oxidative damage through a preconditioning mechanism. The hypothesis is that sulindac can mitigate RPE cell senescence because of its cellular protective properties. To test this, in vitro, an RPE senescence model was established using tert-butyl hydrogen peroxide (TBHP) as an oxidizing agent. The cells were treated with TBHP for 12 hours followed by further incubation for four days. Senescence cells were identified using a Beta-galactosidase assay. The effect of sulindac on senescence was determined by pre-treating cells with varying concentrations of sulindac for 24 h prior to TBHP treatment. Treatment with TBHP induces senescence in the RPE cells under the conditions used. The Preliminary data shows that there was a reduced number of senescent cells in the samples treated with sulindac compared to the control groups. These findings indicate that oxidative damage induces cell senescence, and sulindac can lower the amount of senescence by protecting the RPE cells from oxidative damage. To summarize, the results suggest that sulindac can be used as a therapeutic agent targeting senescence induced by oxidative damage.
  26. Cells. 2022 Apr 28. pii: 1489. [Epub ahead of print]11(9):
      Detection and quantification of senescent cells remain difficult due to variable phenotypes and the absence of highly specific and reliable biomarkers. It is therefore widely accepted to use a combination of multiple markers and cellular characteristics to define senescent cells in vitro. The exact choice of these markers is a subject of ongoing discussion and usually depends on objective reasons such as cell type and treatment conditions, as well as subjective considerations including feasibility and personal experience. This study aims to provide a comprehensive comparison of biomarkers and cellular characteristics used to detect senescence in melanocytic systems. Each marker was assessed in primary human melanocytes that overexpress mutant BRAFV600E, as it is commonly found in melanocytic nevi, and melanoma cells after treatment with the chemotherapeutic agent etoposide. The combined use of these two experimental settings is thought to allow profound conclusions on the choice of senescence biomarkers when working with melanocytic systems. Further, this study supports the development of standardized senescence detection and quantification by providing a comparative analysis that might also be helpful for other cell types and experimental conditions.
    Keywords:  beta-galactosidase; melanocyte; melanoma; senescence
  27. FASEB J. 2022 May;36 Suppl 1
      Pulmonary hypertension (PH) is a fatal pulmonary vascular disease characterized by a sustained elevation of pulmonary arterial pressure. One of the major characteristics of PH is uncontrolled accumulation of pulmonary artery smooth muscle cells (PASMCs) to normally non-muscularized distal pulmonary arteries (PAs). Cellular senescence contributes to aging and lung diseases associated with PH. The aim of this study is to understand the mechanism by which cellular senescence controls vascular remodeling in PH. We have reported that a basic helix-loop-helix transcription factor, TWIST1, in endothelial cells (ECs) mediates hypoxia-induced accumulation of PASMCs to PAs by increasing platelet-derived growth factor (PDGFB) expression. Here we have demonstrated that the levels of senescence markers, p16INK4A and senescence-associated β-galactosidase (SA-β-gal) are higher in ECs isolated from PH patients compared to those from healthy individuals. Analysis of publicly available RNAseq data of PH patient lungs reveals the interaction between TWIST1 and senescence-related genes. The levels of PDGFB upregulated in PH patient-derived ECs are inhibited by knocking down p16INK4A expression or treatment with senolytic reagent ABT-263. Hypoxia-induced accumulation of a-smooth muscle actin (aSMA)-positive cells to the PAs and TWIST1 expression are attenuated in p16INK4Afl/fl -Cdh5(PAC)-CreERT2 mice after tamoxifen induction. These results suggest that EC senescence mediates vascular remodeling in PH through TWIST1 signaling.
  28. Nature. 2022 May 11.
    Keywords:  Ageing; Neurodegeneration; Neuroscience
  29. FASEB J. 2022 May;36 Suppl 1
      BACKGROUND: Reversion of activated hepatic stellate cells (HSCs) to quiescence is likely to be a promising pathway in liver fibrosis regression. The development of senescence in HSCs during alcoholic liver injury may lead to liver compensation against fibrosis. The current study aims to characterize the functional role of miR-34a regulated cellular senescence during alcohol-associated hepatitis.METHODS: Senescence related gene and miR-34a expression was assessed using a PCR Array and/or real-time PCR analysis. Cellular senescence in cultured HSCs were measured by SA-β-gal staining. Senescence mediators were defined in LPS treated hepatic stellate cells in vitro, and in TLR-4 knockout mice or morpholino antisense oligomer against miR-34a (miR-34a Morpho/AS) treated mice with chronic ethanol feeding in vivo.
    RESULTS: We identified that 5 weeks of ethanol feeding significantly increased the total liver histopathology score and miR-34a expression, along with the activation of hepatic stellate cells marked by enhanced α-SMA staining. Treatment of HSCs with LPS (20 ng/ml) for 24 hr significantly increased miR-34a expression, along with the enhanced SA-β-gal activity, up-regulated α-SMA/Collagen A1 expression and increased cellular viability. Silencing of miR-34a decreased LPS-induced activation in HSCs by downregulation of fibrosis markers α-SMA, Col1a1 and TIMP-1, and increased SA-β-gal activity as well as the cellular senescence marker CCl2, whereas silencing of the LPS receptor, TLR4, decreased this marker in the same group of HSCs, suggesting anti-miR-34a reversed LPS-mediated HSC activation through inducing cellular senescence. Furthermore, the expression of miR-34a and verified miR-34a related senescence marker CCl2 were significantly altered in hepatic stellate cells from ethanol-fed mouse liver specimens compared to controls. TLR4 knockout mice and miR-34a Morpho/AS treated mice displayed less sensitivity to alcoholic injury, along with enhanced SA-β-gal activity and CCl2 level in HSCs, and recovered expressions of α-SMA, Col1a1 and TIMP-1.
    SUMMARY AND CONCLUSION: Our results show that miR-34a mediated cellular senescence is essential for the potential reversion from activated to quiescent hepatic stellate cells during alcohol-associated hepatitis. These findings provide new insight into the function of microRNA regulated cellular senescence in human HSCs and increase opportunities for the development of novel treatment paradigms for the management of alcohol-associated liver diseases.
  30. FEBS J. 2022 May 08.
      A new method for senescent cell detection, based on lipofuscin labeling with a fluorescent reporter through a biorthogonal strain-promoted azide-alkyne cycloaddition (SPAAC), is described. The sensing protocol involves a first step where the interaction of lipofuscin with a Sudan Black B derivative containing an azide moiety (SBB-N3) is carried out. In the final step, the azide moiety reacts with a fluorophore containing a cyclooctine ring (BODIPY). The efficacy of this two-step protocol is assessed in senescent melanoma SK-MEL-103 cells, senescent triple-negative breast cancer MDA-MB-231 cells, and senescent WI-38 fibroblasts. In all cases, a clear fluorescence pattern was observed in senescent cells, compared to proliferative cells, only when the SBB-N3-BODIPY probe was formed. Our results provide an alternative tool for the detection of senescent cells, based on an in situ bio-orthogonal reaction for lipofuscin labeling.
    Keywords:  Cellular senescence; Detection; Lipofuscin; Sudan Black B; alkyne-azide cycloaddition
  31. FASEB J. 2022 May;36 Suppl 1
      Aging is a natural process, that is associated with changes in dynamic biological, physiological, environmental, psychological, behavioral, and social processes. In fact, advancing age is the major risk factor for several chronic diseases in humans. During our study of impaired wound healing in aged models, we analyzed skin lysates of young and old mice by LC-MS/MS to identify age-related changes in the proteome. Within the proteins whose levels vary during aging we identified TMPRSS11a, a protein that is overexpressed in aged skin. Transmembrane protease serine 11a (TMPRSS11a, gene ECRG1) is an enzyme, which play a role in cellular senescence and may play a role in SARS-CoV2 infection. This protein has been described at the enzymatic level, but its three-dimensional structure and interactome, also its physiological role in the cell, are still unknown. This protease features three clearly defined domains: Peptidase S1, SEA and cytoplasmic domain. Interestingly, within the structure we have detected two sequences of interest, such as the RGD motif, necessary to interact with integrins and an ITIM sequence involved in immunological activation. Through computational analysis we found that the Peptidase domain contains the hydrophobic pocket that inside it presents the classic catalytic triad (H230, D275 and S371) and next to the catalytic serine there is a RGD motif that meets the characteristics of the canonical motif. Interestingly, using molecular dynamics, docking and proximity ligation assay, we found that the RGD motif is functional and allows direct interaction with integrin β1 and this RGD motif of TMPRSS11a binds directly to the site adjacent to MIDAS in the integrin β1. Finally, we evaluated cellular spreading of HEK293 cells, seeded over a fibronectin matrix, and cell migration through trans well migration assays. And in this way, TMPRSS11a may affect processes such as migration or cell adhesion in an RGD-dependent manner. The expression of this protease could affect processes of vital importance such as wound healing in old animal models. Considering our results, the study of this protein can be used as new therapeutic target of aging.
  32. FASEB J. 2022 May;36 Suppl 1
      Growing evidence shows that expression of non-coding repetitive sequences, or repetitive element (RE) transcripts, increases with age and may be related to biological aging itself. In fact, RE transcript levels can be used to accurately predict age, and they are reduced by healthy aging interventions. However, the exact mechanistic links between RE dysregulation and aging are unknown. Some evidence indicates that RE transcripts may stimulate age-related inflammation (via formation of RE-derived double-stranded RNA, an innate immune activator) but there is currently no evidence of this in human subjects. Therefore, we determined if serum interleukin 6 (IL-6), a key circulating marker of inflammation, was associated with RE transcript expression (assessed via RNA-seq) in 92 healthy, middle aged to older adults (64 ± 7 years, 47M/ 45F). We found that serum IL-6 was positively related to expression of every major type of RE (LINEs, SINEs, DNA transposons, LTRs, and Satellites; mean r value = 0.4, p < 0.0005). To examine transcriptome/gene expression differences associated with high vs. low serum IL-6, we analyzed differential gene expression in subjects with the highest vs. lowest IL-6 levels (6 per group, age/sex-matched). We then performed gene ontology analysis to determine biological processes associated with differentially expressed genes. Interestingly, we found that higher IL-6 was associated with reduced gene expression signatures of immune function and increased gene expression signatures related to cell death and differentiation. To assess a potential upstream link between RE expression and inflammation, we looked for correlations among RE and innate immune sensor transcript levels. We found a positive relationship between RE expression and levels of IFIH1, the gene that encodes MDA5, a proinflammatory sensor of double-stranded RNA (p < 0.0005, mean r value = 0.5). MDA5 expression correlated weakly with LINE expression (r = 0.36), but more strongly with satellite and LTR expression (r = 0.66), suggesting that these RE types may be involved in age-associated inflammation. These are the first data to show a relationship between IL-6 and global RE expression in older adults, and to characterize the possible mechanisms underlying this relationship.
  33. Sci Rep. 2022 May 11. 12(1): 7766
      Senescence affects various aspects of sleep, and it remains unclear how sleep-related neuronal network activity is altered by senescence. Here, we recorded local field potential signals from multiple brain regions covering the forebrain in young (10-week-old) and aged (2-year-old) mice. Interregional LFP correlations across these brain regions could not detect pronounced differences between awake and sleep states in both young and aged mice. Multivariate analyses with machine learning algorithms with uniform manifold approximation and projection and robust continuous clustering demonstrated that LFP correlational patterns at multiple frequency bands, ranging from delta to high gamma bands, in aged mice less represented awake/sleep states than those in young mice. By housing aged mice in an enriched environment, the LFP patterns were changed to more precisely represent awake/sleep states. Our results demonstrate senescence-induced changes in neuronal activity at the network level and provide insight into the prevention of pathological symptoms associated with sleep disturbance in senescence.
  34. FASEB J. 2022 May;36 Suppl 1
      INTRODUCTION: The nuclear factor erythroid 2-related factor 2 (Nrf2) is a master transcription factor governing hundreds of genes coding proteins involved in anti-oxidation, anti-inflammation, detoxification, and metabolism. Using a model of tissue-specific deletion of the gene for Kelch Like ECH Associated Protein 1 (Keap1), an endogenous inhibitor of Nrf2, we previously found that Nrf2 overexpression upregulates over 100 proteins in skeletal muscle (SkM), which results in activation of endogenous antioxidant defenses and the enhancement of exercise capacity in adult mice. In addition, by employing a SkM-reporter mouse, where SkM exclusively expresses GFP while non-muscle tissues express td-Tomato, we demonstrated that SkM-derived extracellular vesicles (EVs) can be transferred from SkM to remote non-SkM tissues, including cardiomyocytes. Given the critical role of oxidative stress in the pathogenesis of sarcopenia and cardiac aging, we hypothesized that Keap1 KO can protect against aging-associated myopathy not only in SkM but also in myocardium via EV-mediated inter-organ antioxidant protein communication.MATERIALS & METHODS: Experiments were carried out in 43 male iMS-Keap1flox/flox (i.e. Keap1 SkM specific) mice assigned to 4 groups: young-WT (11), young-Keap1-KO (12), aged-WT (11), and aged-Keap1-KO (9). Keap1 KO was induced by intraperitoneal injection of tamoxifen at 3 months while the WT group received sunflower oil (vehicle) at the same age. At the age of 6 months in young groups and 27 months in aged groups, mice a maximal exercise tolerance test (treadmill) and echocardiography to evaluate cardiac function.
    RESULTS: We found that, aged-WT displayed significantly shorter running distance than young-WT (280.2 ± 35.1 vs 531.6 ± 38.4 m, p < 0.001). This was improved in aged-Keap1-KO mice (508.2 ± 66.8 m, p < 0.01 vs aged WT). Compared with young-WT, aged-WT displayed significantly reduced SkM weights of soleus (0.0128 ± 0.0011 vs 0.0182 ± 0.0021 g, p < 0.05) and tibialis anterior (0.0627 ± 0.0093 vs 0.0937 ± 0.0101 g, p < 0.005), which were ameliorated in aged-Keap1-KO mice. Echocardiography indicated that aged-WT mice have lower ejection fraction (EF), longer isovolumic relaxation time (IVRT), and higher myocardial performance index (MPI) than young-WT (EF: 60.3 ± 3.1 vs 68.1 ± 1.3 %, p < 0.05; IVRT: 22.6 ± 1.5 vs 16.9 ± 0.5 ms, p < 0.01; MPI: 0.71 ± 0.03 vs 0.55 ± 0.02, p < 0.001). These aging-associated cardiac dysfunctions were partially alleviated in aged-Keap1-KO (EF: 65.6 ± 0.65 %, p = 0.073; IVRT: 16.4 ± 1.5 ms, p < 0.01; MPI: 0.57 ± 0.03, p < 0.001 vs aged WT). In addition, aged-WT mice exhibited attenuated responses to intraperitoneal injection of the ß-1 agonist, dobutamine. EF, cardiac output, stroke volume, and diastolic volume, were significantly improved in aged-Keap1-KO mice (p < 0.05 vs aged-WT).
    CONCLUSIONS: Our data suggest that a life-long activation of SkM Nrf2 not only attenuates aging-associated SkM dysfunction but also improves cardiac aging parameters. The later effect, we propose is mediated by transference of Nrf2-dependent upregulated antioxidant enzymes in the Keap1-deficient SkM to the myocardium through SkM-derived EVs.
  35. FASEB J. 2022 May;36 Suppl 1
      Neuroinflammation is a central mechanism of brain aging and neurodegenerative disease, but why neuroinflammation develops with aging is incompletely understood. Adenosine deaminase RNA specific (ADAR) proteins are potential suppressors of neuroinflammation through their adenosine-to-inosine (A-to-I) editing and subsequent inhibition of double-stranded RNA (dsRNA, an innate immune activator). However, the role of ADARs in brain aging and/or neurodegenerative disease (e.g., Alzheimer's disease, AD) is unclear. To address this, we looked for age-related changes in ADAR expression and function (indicated by A-to-I editing) in multiple, brain-specific transcriptomic datasets. We found evidence of age-related declines in ADAR expression in: 1) astrocytes (glial cells linked with immune/inflammatory signaling in aging and AD) in old vs. young mice; 2) induced astrocytes from old vs. young human subjects; and 3) brain tissue of AD patients vs. healthy age-matched controls. We also found declines in A-to-I editing related to reduced ADAR expression, suggesting that ADAR and ADAR activity decline with age and disease. We followed up on these observations via in vitro experiments in primary human astrocytes, in which we suppressed ADAR via siRNA knockdown or with an ADAR inhibitor (8-Azaadenosine). These treatments increased levels of the proinflammatory cytokine interleukin 6 (IL-6) and intercellular adhesion molecule 1 (ICAM-1, a marker of pro-inflammatory astrocytes), and this was associated with accumulation/changes in the cellular distribution of dsRNA (assessed via immunofluorescence). Preliminary data showed that ADAR knockdown tended to increased levels of protein kinase R (PKR, a key pro-inflammatory dsRNA sensor), and that dsRNA colocalizes with PKR. To identify related mechanisms, we are currently performing RNA-seq to profile whole-transcriptome changes associated with ADAR inhibition in astrocytes. Taken together, our data suggest that ADAR may be an important modulator of neuroinflammation in aging, and ultimately neurodegenerative diseases.
  36. Int J Mol Sci. 2022 Apr 27. pii: 4843. [Epub ahead of print]23(9):
      An increased life span and accompanying nutritional affluency have led to a rapid increase in diseases associated with aging, such as obesity and type 2 diabetes, imposing a tremendous economic and health burden on society. Pancreatic β-cells are crucial for controlling glucose homeostasis by properly producing and secreting the glucose-lowering hormone insulin, and the dysfunction of β-cells determines the outcomes for both type 1 and type 2 diabetes. As the native structure of insulin is formed within the endoplasmic reticulum (ER), ER homeostasis should be appropriately maintained to allow for the proper metabolic homeostasis and functioning of β-cells. Recent studies have found that cellular senescence is critically linked with cellular stresses, including ER stress, oxidative stress, and mitochondrial stress. These studies implied that β-cell senescence is caused by ER stress and other cellular stresses and contributes to β-cells' dysfunction and the impairment of glucose homeostasis. This review documents and discusses the current understanding of cellular senescence, β-cell function, ER stress, its associated signaling mechanism (unfolded protein response), and the effect of ER stress on β-cell senescence and dysfunction.
    Keywords:  ER stress; cellular senescence; endoplasmic reticulum; insulin; islet amyloid polypeptide; pancreatic beta cell; type 2 diabetes
  37. Geroscience. 2022 May 09.
      Cytochrome b5 reductase 3 (CYB5R3) overexpression activates respiratory metabolism and exerts prolongevity effects in transgenic mice, mimicking some of the salutary effects of calorie restriction. The aim of our study was to understand how CYB5R3 overexpression targets key pathways that modulate the rate of aging in skeletal muscle, a postmitotic tissue with a greater contribution to resting energy expenditure. Mitochondrial function, autophagy and mitophagy markers were evaluated in mouse hind limb skeletal muscles from young-adult (7 months old) and old (24 months old) males of wild-type and CYB5R3-overexpressing genotypes. Ultrastructure of subsarcolemmal and intermyofibrillar mitochondria was studied by electron microscopy in red gastrocnemius. CYB5R3, which was efficiently overexpressed and targeted to skeletal muscle mitochondria regardless of age, increased the abundance of complexes I, II, and IV in old mice and prevented the age-related decrease of complexes I, III, IV, and V and the mitofusin MFN-2. ATP was significantly decreased by aging, which was prevented by CYB5R3 overexpression. Coenzyme Q and the mitochondrial biogenesis markers TFAM and NRF-1 were also significantly diminished by aging, but CYB5R3 overexpression did not protect against these declines. Both aging and CYB5R3 overexpression upregulated SIRT3 and the mitochondrial fission markers FIS1 and DRP-1, although with different outcomes on mitochondrial ultrastructure: old wild-type mice exhibited mitochondrial fragmentation whereas CYB5R3 overexpression increased mitochondrial size in old transgenic mice concomitant with an improvement of autophagic recycling. Interventions aimed at stimulating CYB5R3 could represent a valuable strategy to counteract the deleterious effects of aging in skeletal muscle.
    Keywords:  Aging; Autophagy; Cytochrome b 5 reductase; Mitochondria; Skeletal muscle
  38. FASEB J. 2022 May;36 Suppl 1
      PURPOSE: Retinal pigmented epithelium (RPE) dysfunction is associated with age-related macular degeneration (AMD), the leading cause of vision loss in the elderly in the western world. A better understanding of RPE-related signaling pathways is the key to developing therapies and potential drug treatments for the early stages of AMD. Nuclear receptors are master regulators of cellular processes including lipid metabolic dysregulation, apoptosis, and inflammation, pathogenic pathways associated with AMD development and progression. Previously we found that orphan nuclear receptor member 1(NR4A1) or NUR77 is expressed by human RPE. The role of NUR77 in the eye or RPE-mediated signaling pathways, however is not known. Given NUR77's purported role in regulating inflammation, angiogenesis, and apoptosis in a number of body tissues, herein we investigated the role of NUR77 in RPE as a function of age.METHODS: Total RNA was extracted from RPE/choroid fractions of young and old mouse eyes, RPE/choroid from human donors, and human primary RPE cultures harvested from young and old eye tissue (ages 18-93). Expression of NUR77/Nurr77 and its target genes were measured by qRT-PCR. Cellular bioenergetics of young versus old RPE and NUR77 overexpressing RPE were measured using the Seahorse XF Real-Time ATP Rate Assay. RPE cells overexpressing NUR77 were analyzed for expression of senescence-related and epithelial-mesenchymal transition (EMT) markers. To investigate the function of Nur77, in vivo, ocular imaging and retinal function assessments of 4-8 month old Nur77-null mice was done. Ex vivo, ocular morphology was examined. Statistical methods for data analysis included two-tailed Student's t-test and two-way ANOVA. Values were considered statistically significant at p < 0.05.
    RESULTS: Nur77/NUR77 expression in mouse and human RPE decreases with age. Overexpression of NUR77 (induction of the 'young phenotype') in RPE cells isolated from old donors results in a decrease in expression of the senescence associated genes p21 and p16, while low levels of NUR77 were associated with elevated senescence. Expression of the metabolic markers COX III, pyruvate kinase M1 and 2were differentially regulated in the RPE/choroid fractions from old versus young mice. Seahorse assays revealed that overexpression of NUR77 in RPE cells from young but not old donors resulted in a metabolic shift from glycolysis towards mitochondrial respiration. In the absence of Nur77, enhanced expression of markers of inflammation (CCL2, TNFa), oxidative stress (HMOX1, SOD1), metabolism (COXIII, PKM2) and EMT (N-cadherin, Snail 2, ZEB1, FN1, Vimentin) was observed in the mouse RPE/choroid. Initial assessments of ocular imaging revealed the presence of hypopigmented regions within the posterior pole of Nur77-null mice at 4 months of age.
    CONCLUSIONS: Our preliminary results indicate an age-dependent role of NUR77 in balancing a metabolic switch towards mitochondrial respiration in the RPE. Studies are ongoing to further dissect the NUR77-mediated signaling pathways in aging and ocular disease, and in determining if NUR77 overexpression may serve as potential therapy in diseases wherein RPE is compromised.
  39. FASEB J. 2022 May;36 Suppl 1
      Bone fragility increases with age as the result of the concurrent decline of bone mass, quality, and mechanosensitivity. While the coordinated decline of these behaviors remains unexplained, the role of osteocytes in each of these processes, and the age-related degeneration of the lacunocanalicular network (LCN) in which they reside, implicate osteocytes in bone aging. Using a combination of imaging and computational modeling, we identify canalicular pruning as a driver behind declining bone functionality with age. Furthermore, these analyses predict novel opportunities to restore function to aged osteocyte networks through expansion of the pericellular space.
  40. FASEB J. 2022 May;36 Suppl 1
      Age is a risk factor for many diseases including idiopathic pulmonary fibrosis (IPF) by affecting wound healing through abnormal fibrogenesis. Common cellular and molecular traits associated with aging can be found in senescent cells, which may occur throughout the lifespan playing physiological roles during normal development or limiting the proliferation of damaged and aged cells. In this study we investigated in smoking mice whether the senescence of lung cells, triggered by the oxidative stress response, is associated with molecular pathways that can influence lung fibrogenesis and contribute to the remodeling of the fibrotic tissue characterizing some phenotypes of COPD in humans. Among the complex mixture of hundreds senescence secreted factors that include proinflammatory cytokines, chemokines, growth factors, and proteases, we selected important factors involved in differentiation of myofibroblasts, their dedifferentiation and apoptosis, as well as in their metabolic function. The expression of these factors and the amount of matrix deposition was analyzed after chronic cigarette smoke (CS) exposure of C57 Bl/6 and DBA/2 mice sensitive to oxidative, in different lung compartmentscharacterized by a fibrotic remodeling using histochemistry or immunohistochemistry. Unlike C57 Bl/6, DBA/2 mice at 4 months of exposure show marked positivity for 8-OHdG, a marker of oxidative damage, in subpleural areas, in central parts of the lung parenchyma, on airways and on cells of the fibro-muscular layer. In these areas, a marked positivity is observed for fibrogenic cytokines such as TGF-β, PDGF-b and CTGF, and for other factors such as the p16ink4A senescence marker, and proliferation markers PCNA and Ki67. In DBA/2 mice, these compartments are characterized by a progressive fibrous remodeling at various experimental time points from 5 months of CS exposure onwards as demonstrated by Masson's trichrome staining. Fibroproliferative foci are present in sub-pleural areas, in peripheral areas of airways, and in central parts of the lung parenchyma. They are characterized by increasing number of α-SMA positive myofibroblasts, which express the senescence marker p16ink4A and the transcription factor MyoD that plays a crucial role in myoblast proliferation and differentiation. MyoD induces cell cycle arrest, promotes cellular terminal differentiation of fibroblasts into myofibroblasts, and in senescent myofibroblasts it has been reported to determine resistance to apoptosis and opposes their dedifferentiation. All these factors contribute to the development in smoking DBA/2 mice of lung lesions, which are like those seen in patients with "Combined Fibrosis/Emphysema Syndrome." Despite consistent levels of TGF-β, PDGF-b and CTGF, C57 Bl/6 mice develop after chronic CS exposure fibrotic remodeling at lower extent. Of interest, a very low expression of factors indicative of senescence (p16ink4A and MyoD) and cell proliferation (PCNA and Ki67) is observed. Our results suggest that apoptosis, senescence, and proliferation induced at different rate by fibrotic cytokines and senescence associated secretory factors play important role in early or late appearance of fibrotic remodeling of lung and airways exposed to CS.
  41. FASEB J. 2022 May;36 Suppl 1
      BACKGROUND: The accumulation of senescent cells is a hallmark of aging in the skeletal muscle which causes reactive oxygen species (ROS) and mitochondrial dysfunction. p21 is one of the major regulators and most recognized cellular markers for senescent cells. Recent evidence demonstrated that clearance of p21high cells enhances muscle function including grip strength, hanging endurance, and maximal walking speed in mice. However, it is still unclear how the muscle performance enhances through the clearance of p21high cells. One of the potential mechanisms is mitochondrial function and/or mitochondria-derived ROS. Therefore, this study investigated the linkage between mitochondria and p21high cells in aging or high fat diet-induced muscle dysfunction.METHODS: Five p21-Cre/+; +/+ (P) and p21-Cre/+; DTA/+ (PD) obese mice fed by high fat diet were administrated with tamoxifen for twice. Five P lean mice fed by normal chow were adopted as normal control. We previously demonstrated that p21high cells accumulate in obese P mice, and can be eliminated in obese PD mice by tamoxifen treatment. Mitochondrial respiration was measured, by high-resolution respirometry, in permeabilized muscle fibers from the soleus muscle. Mitochondria-derive ROS production was determined using Amplex Red assays. One-way ANOVAs with Bonferroni post-hoc were used to determine differences between groups (P < 0.05).
    RESULTS: Reductions in complex I + II state 3 respiration were observed in p21-Cre mice with high fat diet but the clearance of p21high cells using tamoxifen enhanced complex I + II state 3 respiration (Lean P: 35.2 ± 3.13 pmol·s-1 ·mg-1 ; Obese P: 17.45 ± 2.85 pmol·s-1 ·mg-1 ; Obese PD: 30.04 ± 3.19 pmol·s-1 ·mg-1 ; P < 0.05). State 4 respiration did not differ between groups (P > 0.05). Respiratory Control Ratio (RCR), defined as respiration in state 3 divided by respiration in state 4, significantly decreased in high fat diet group but clearing p21high cells restored respiratory function (Lean P: 4.20 ± 1.18; Obese P: 2.21 ± 1.11; Obese PD: 3.85 ± 1.60; P<0.05). Also, high fat diet increased mitochondrial ROS production, but tamoxifen treatment attenuated ROS production (Lean P: 10.16 ± 0.41 pmol·s-1 ·mg-1 ; Obese P: 26.83 ± 0.54 pmol·s-1 ·mg-1 ; Obese PD: 12.23 ± 0.54 pmol·s-1 ·mg-1 ; P<0.05).
    CONCLUSION: These results demonstrated that p21high cells may play a causal role in mitochondrial dysfunction and ROS emission in the skeletal muscle with obesity and other chronic diseases. Therefore, targeting p21high cells may enhance muscle performance through decreasing mitochondria-derived ROS and enhancing mitochondrial function.
  42. FASEB J. 2022 May;36 Suppl 1
      BACKGROUND: KDM6A mediates the removal of repressive trimethylation from H3K27me3 to activate target gene expression. It is not known, however, whether KDM6A plays a role in the regulation of cardiac aging. We hypothesized that cardiac-specific KDM6A knockout accelerates cardiac aging.METHODS AND RESULTS: Aging decreased histone demethylase KDM6A expression and activity in human cardiomyocytes. Downregulation of KDM6A expression was also found in cardiomyocytes of aged mice. The cardiomyocytes-specific KDM6A knockout (Myh6-KDM6A cKO) mouse model was created by using Cre-LoxP system. KDM6A deletion didn't cause overt cardiac remodeling and dysfunction in normal condition. However, cardiomyocytes-specific conditional knockout of KDM6A exaggerated ISO-induced cardiac dysfunction and cardiac remodeling. Moreover, Myh6-KDM6A cKO mice showed early cardiac aging at 23-month-old, while control mice didn't display cardiac aging until 27-month-old. Therefore, KDM6A deletion accelerated cardiac aging. ChiP-Seq analysis revealed candidate transcription factors participating in KDM6A cKO-induced cardiac aging. In particular, HoxC4 was down-regulated in the cardiomyocytes of Myh6-KDM6A cKO mice. Knockdown of HoxC4 gene in H9c2 cardiomyoblast cell line caused ER stress. The ER stress further resulted in oxidative stress and cardiomyocytes apoptosis in cardiomyocytes-specific conditional knockout of KDM6A mice.
    CONCLUSIONS: Cardiomyocytes-specific conditional knockout of KDM6A causes cardiac aging through induction of HoxC4-mediated ER stress. These observations suggest KDM6A a potential therapeutic target for cardiac aging.
  43. FASEB J. 2022 May;36 Suppl 1
      BACKGROUND AND OBJECTIVE: Sarcopenia is defined as the loss of skeletal muscle mass and function due to age, which is a major concern for life long health and wellbeing in elderly population. Age-associated decline in the performance of muscle stem cells (MuSC) contributes to the deterioration of skeletal muscle during aging. Using small molecule Givinostat and CHIR99021 we previously demonstrated the generation of muscle progenitor cells (Givi-MPC) of human Induced pluripotent stem cells (iPSC) from multiple cell lines. We found that extracellular vesicles (EVs) from these MPC have a specific cargo compared with human myoblasts. Here, we investigated whether these EVs can rejuvenate the aging dysfunctional muscle stem cells.METHODS AND RESULTS: Using size exclusion chromatography EVs were isolated and purified from iPSC derived Givi-MPC and primary human myoblasts. The EVs were visualized by transmission electron microscopy. Next, we isolated primary MuSC from 25 months old aged C57/B6 mice, which were confirmed by the expression of myogenic transcription factor, Pax7. We found the aged MuSC pretreated with EVs from MPC promoted clonal expansion and restored myogenic potential in vitro with more myotubes formed after 72h differentiation in medium with 2% horse serum compared with PBS pretreated MuSC (MuSC (Fusion index: 40.3±5.2% vs.15.0± 2.1%, n=3).). To test the regenerative potential of the EVs on aged MuSC, 8 weeks old mdx/scid mice (dystrophin deficient mice) were irradiated to destroy the host MuSC and then injured their tibialis anterior (TA) muscle with cardiotoxin (CTX). 24 hours after injury, we transplanted the aging MuSC (5000 cells) either with EV or PBS pretreatment into TA muscles, respectively. 5 days after transplantation, the EVs pretreated MuSC increased muscle regeneration with muscle fibers positive for embryonic myosin heavy chain compared to PBS pretreated MuSC. One month later, we found transplantation of EVs pretreated MuSC restored dystrophin expression in these mdx mice. We further tested the effects of EVs pretreatment on MuSC in 25 months old mice following CTX injury. Consistent with the results in mdx mice, pretreatment of mice with EVs from Givi-MPC showed enhanced regeneration (6-fold increase in number of new muscle cells with central nuclei) in CTX injured aged muscle accompanied by an increase of Pax7 positive MuSC compared with PBS treated mice or treatment with EVs from human myoblasts (number of Pax7 positive MuSC: EVs-Givi-MPC 10±5/mm2; PBS 2±1/mm2; EVs-human myoblast 6±2/mm2, n=3).
    CONCLUSION: The regenerative potential of aging MuSC is restored with the treatment of EVs from Givi-MPC allowing stem cell renewal and differentiation upon activation.
  44. FASEB J. 2022 May;36 Suppl 1
      Aging is accompanied by striking changes in chromatin and gene expression across cell types and species. Yet, how chromatin landscapes change with age and regulate transcription, and how epigenomic changes in turn influence aging in response to external or internal cues, is largely unknown. In addition, accumulating evidence indicates that sex hormones play a key role in driving aspects of cellular and molecular sex-dimorphism. In parallel to sex hormones, karyotypic sex (i.e. XX vs. XY) is also likely to have important impact outside of gonadal sex determination. A key compartment whose activity can be actively modulated by sex-dimorphic mechanisms throughout life is the immune system, whose function declines sharply with aging and may be actively modulated by sex. Indeed, aspects of the immune responses differ between sexes, with a more robust immune response in females and an increased susceptibility to infection in males. Thus, our main cell models of study are key components of the innate immune system and the inflammatory response: macrophages, which accomplish key tasks such as phagocytosis, antigen presentation and cytokine production, and neutrophils, the most abundant leukocyte type serving as a "first line of defense" against infection. Excitingly, we and others have observed strong sex-related differences in the transcriptional and functional phenotypes of these cells and have observed sex-dimorphic "omic" trajectories for these cells with aging. Based on our data and published literature, it is likely that mechanisms involving both gonadal hormones and sex chromosomes may fine-tune different aspects of immunity and, thus, overall health and lifespan.
  45. FASEB J. 2022 May;36 Suppl 1
      An increased presence of the oxidised DNA base lesion 8-oxo-7,8-dihydroguanine (8-oxoG) is seen in muscles of old mice. 8-oxoG is repaired by the 8-oxoguanine DNA glycosylase-1 (OGG1)-initiated DNA base excision repair pathway, a process proposed to lead to activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB). We hypothesised that chronic oxidative DNA damage in muscles of old mice leads to the chronic increase in activation of NF-kB by OGG1 and the subsequent production of inflammatory cytokines by skeletal muscle, contributing to age-related deterioration of muscle function. An in vitro model of oxidative DNA damage was established by treating C2C12 myoblasts with H2O2. DNA damage repair responses including 8-oxoG, OGG1, PARP-1, NF-kB activation and cytokine/chemokine gene expression and release by muscle cells were determined using Luminex multiplex analysis. The effect of TH5487 (5µM), a selective OGG1 inhibitor on NF-kB activation and cytokine/chemokine production was determined. The role of OGG1 in the chronic production of cytokines/chemokines by isolated muscle fibres of adult mice treated with H2O2 and of old mice was examined. Cell viability was maintained in myoblasts treated with 50µM H2O2 but Ogg1 mRNA levels were significantly increased as was acetylation of OGG1, PARP-1 and cleaved PARP-1 protein levels. Activation of NF-κB was evident and accompanied by an increased expression and release of a number of cytokines/chemokines (3.7 fold, 4.2 fold, 3 fold increases in IL-6 TNF-a and CXCL1 mRNA respectively). The H2O2-mediated increase in NF-kB activation and increase in cytokine/chemokine mRNA levels were normalized when cells were pre-treated with TH5487 prior to treatment with H2O2. Experiments using isolated muscle fibers from adult mice demonstrated an increase in NF-κB activation following treatment with H2O2. This H2O2-mediated increase in NF-kB DNA binding activity was significantly decreased in muscle fibers pre-treated with TH5487. Isolated muscle fibers from old compared with adult mice showed an increase in cytokine/chemokine release which was also reduced by treatment with TH5487. Increases in IL-6, CCL2,CXCL1,CCL7,CCL27,CXCL5,CCL11,CXCL16 and CXCL-12 were seen in the media of fibers from adult mice following treatment with H2O2 and most of these were then decreased to normal values by pre-treatment with TH5487. Isolated muscle fibers from old mice showed a significant increase in IL-6, CXCL1,CCL2,CCL7, of which all were reduced following treatment with TH5487, suggesting that activation of the OGG1 pathway may be responsible, at least in part for the increased production of cytokines/chemokines by muscle of old mice. Data on proteomic and mRNA analyses of muscles from adult and old mice for evidence of changes in DNA repair pathways with age will also be presented. A computational model of DNA damage repair is being designed to identify DNA damage repair process therapeutic targets to modify activation of NF-κB in muscles of old mice.
  46. Int J Neurosci. 2022 May 13. 1-26
      Sirtuins are Nicotinamide Adenine Dinucleotide (NAD+) dependent class ІΙΙ histone deacetylases enzymes (HDACs) present from lower to higher organisms such as bacteria (Sulfolobus solfataricus L. major), yeasts (Saccharomyces cerevisiae), nematodes (Caenorhabditis elegans), fruit flies (Drosophila melanogaster), humans (Homo sapiens sapiens), even in plants such as rice (Oryza sativa), thale cress (Arabidopsis thaliana), vine (Vitis vinifera L.) tomato (Solanum lycopersicum). Sirtuins play an important role in the regulation of various vital cellular functions during metabolism and ageing. It also plays a neuroprotective role by modulating several biological pathways such as apoptosis, DNA repair, protein aggregation, and inflammatory processes associated with ageing and neurodegenerative diseases. In this review, we have presented an updated Sirtuins and its role in ageing and age-related neurodegenerative diseases (NDDs). Further, this review also describes the therapeutic potential of Sirtuins and the use of Sirtuins inhibitor/activator for altering the NDDs disease pathology.
    Keywords:   Sirtuins ; ageing; human; neurodegenerative diseases
  47. FEBS Lett. 2022 May 09.
      The advancement of medical technology has led to an increase in life expectancy but also to a rise in ageing-related diseases. Ageing promotes metabolic disorders, in turn affecting cardiovascular health. Derailment of biological processes in pancreas, liver, adipose tissue, and skeletal muscle impairs glucose and lipid metabolism as well as mitochondrial function, triggering the development of diabetes and lipid-related disorders that inflict damage on cardiac and vascular tissues. Long non-coding RNAs (lncRNAs) regulate a wide range of biological process and are one of the key factors controlling metabolism and mitochondria. Here, we discuss the versatile function of lncRNAs involved in the metabolic regulation of glucose and lipid, and mitochondrial function, and how the dysregulation of lncRNAs induces the development of various metabolic disorders and their cardiovascular consequences.
    Keywords:  LncRNA; ageing; cardiovascular system; glucose and lipid metabolism; mitochondria
  48. Nature. 2022 May 11.
      Recent understanding of how the systemic environment shapes the brain throughout life has led to numerous intervention strategies to slow brain ageing1-3. Cerebrospinal fluid (CSF) makes up the immediate environment of brain cells, providing them with nourishing compounds4,5. We discovered that infusing young CSF directly into aged brains improves memory function. Unbiased transcriptome analysis of the hippocampus identified oligodendrocytes to be most responsive to this rejuvenated CSF environment. We further showed that young CSF boosts oligodendrocyte progenitor cell (OPC) proliferation and differentiation in the aged hippocampus and in primary OPC cultures. Using SLAMseq to metabolically label nascent mRNA, we identified serum response factor (SRF), a transcription factor that drives actin cytoskeleton rearrangement, as a mediator of OPC proliferation following exposure to young CSF. With age, SRF expression decreases in hippocampal OPCs, and the pathway is induced by acute injection with young CSF. We screened for potential SRF activators in CSF and found that fibroblast growth factor 17 (Fgf17) infusion is sufficient to induce OPC proliferation and long-term memory consolidation in aged mice while Fgf17 blockade impairs cognition in young mice. These findings demonstrate the rejuvenating power of young CSF and identify Fgf17 as a key target to restore oligodendrocyte function in the ageing brain.
  49. FASEB J. 2022 May;36 Suppl 1
      BACKGROUND: Advancing of age decreases the barrier properties of intestinal epithelium which leads to sterile systemic inflammation. Chronic low-grade chronic inflammation is an underlying cause for the age-related comorbidities such as cardiovascular disorders. Renin Angiotensin System (RAS) plays an important role in the homeostasis of cardiovascular functions however, the role of RAS in the aging gut is poorly studied. The protective axis of RAS, which constitutes Angiotensin converting enzyme-2 (ACE2)/ Angiotensin-(1-7) (Ang-(1-7))/Mas receptor (MasR) is known to be counter-regulatory to the classical axis, ACE/Ang II/AT1 receptor (AT1R). This study tested the hypothesis that leaky gut with aging is associated with ACE2/ACE imbalance and that activation of MasR with Ang-(1-7) would restore gut barrier integrity and ameliorates systemic inflammation.METHODS: Study was carried out in Young (3-4 months) and Old (20-24 months) male mice. ACE and ACE2 enzyme activities were evaluated in the colon. Gene and protein expressions of RAS components were determined by western blotting. Ang-(1-7) was administered by subcutaneous osmotic pump (1 µg/Kg/min) for four weeks and the gut permeability was evaluated by using FITC-dextran. Colon wall integrity was evaluated by immunohistochemistry of claudin 1 and occludin. Zonulin-1, IL-6, and TNFα were analyzed in the plasma. A779, MasR antagonist, was administered by an osmotic pump in selected experiments (1 µg/Kg/min, subcutaneous).
    RESULTS: ACE2 protein and activity were decreased in Old group while that of ACE were increased (n=6) (n=8) in the colon of the Old mice compared to the Young. Protein expression of both AT1R and MasR was higher while that of AT2R was lower in the Old (P<0.05, n=8). Protein expression of ACE2, ACE AT1R and MasR in the Old colons were restored to Young levels by treatment with Ang-(1-7). Gut permeability was higher in Old mice (P<0.01 vs Young, n=6) that was reversed by Ang-(1-7). The beneficial effect of Ang-(1-7) was abolished by concurrent administration of A779. Hematoxylin & Eosin staining revealed that aging was associated with atrophied villi in the gut wall that was normalized by Ang-(1-7). Gene expression of claudin 1 and occludin were lower in the Old colon wall that were restored by Ang-(1-7) and similar observations were made by immunohistochemistry. Importantly, plasma levels of Zonulin-1, (P<0.01) IL-6 and TNFα (P<0.05) (n=8) were higher in the Old group compared to the Young, which were lowered by Ang-(1-7) (n=5).
    CONCLUSION: These results suggest that activation of MasR ameliorates aging-associated systemic inflammation by restoring gut barrier integrity, which at least in part contributes to the cardiovascular-protective functions.
  50. FASEB J. 2022 May;36 Suppl 1
      Previously we have found that T cells contribute to age-related large artery stiffness and impairments in endothelium dependent dilation. The thymus is an immunological organ that is responsible for the generation of new naïve T cells. A hallmark of T cell aging is a phenotypic shift from a naïve to a memory phenotype. In this study, we sought to determine 1) whether early life thymectomy at 3wks of age would induce an aged (memory) T cell phenotype and 2) that these alterations in T cell phenotype will result in arterial dysfunction in otherwise middle-aged mice. This study was approved by the institutional animal care and use committee at the University of Texas Arlington. Male C57BL6 mice underwent thymectomy (thymex; n=7-17) at 3wks of age or left with their thymus intact (control; n=7-17). At 9 months of age, doppler pulse wave velocity was used to assess large artery stiffness and following euthanasia, splenic, aortic, and mesenteric T cell phenotype was assessed using flow-cytometry and T cell proliferation was assessed in vitro. To assess endothelium-dependent dilation, second order mesenteric arteries were gently cleared of adipose and connective tissue and cannulated on the stage of a pressure myograph. Group differences were assessed by independent samples t-test or repeated measures ANOVA and Bonferroni post-hoc test. Data are presented as mean±SEM. Pulse wave velocity indicated that the thymex mice had significantly higher aortic stiffness (304.1±12.2 cm/s) compared to controls (270.2±13.8 cm/s; p=0.05). Flow cytometry results (expressed as thymex vs control) demonstrate that the spleen (CD4:79.4±5, 55.4±7.6 %memory; p=0.01; CD8: 72.2±2.4, 44.8±6.7 %memory; p=0.0008), aorta (CD4: 63.4±5.2, 34.6±6.5 %memory; p=0.001; CD8: 64.5±4, 30.6±2.4 %memory; p=<0.0001), and mesentery (CD4: 68.3±5.6,44.2±6.5 %memory; p=0.006; CD8: 47.1±6.1, 31.6±5.7 %memory; p=0.04) all exhibited a greater proportion (relative to all immune cells) of memory CD4+ (helper T) and CD8+ (cytotoxic T) T cells in the thymex animals compared to controls which is consistent with chronological aging. The after 96 hours in culture cell proliferation was blunted in both the CD4+ (45142±7214.4 cells) and CD8+ (50285.7±7870.7 cells) cells from the thymex mice compared to the control mice (CD4: 75428.6±3652.5 cells; p=0.0023; CD8: 81142.9±2756.9 cells; p=0.0025), which is also consistent with an aged T cell phenotype. Finally, the thymex mice exhibited significantly blunted endothelium dependent dilation in response to 10-4 --M acetylcholine (46.1±6.99 % dilation) compared to controls (71.5±5%; p=0.005). Assessing dilation in the presence of L-NAME revealed a significant blunting of nitric oxide bioavailability in arteries from thymex mice (33.91±8.2% NO dependent dilation) compared to controls (55.7±8.5%; p=0.05). In conclusion, these results indicate that early-life thymectomy results in T cell aging as well as arterial dysfunction in otherwise middle-aged mice. These observations suggest that aged T cells alone can drive age-related dysfunction independent of the age of the arteries.
  51. FASEB J. 2022 May;36 Suppl 1
      This study characterized long-term changes in cardiac structure and function in a high-fat diet/streptozotocin mouse model of aging and type 2 diabetes mellitus (T2D) and how the intersection of both conditions alters plasma metabolomics. Furthermore, we wanted to assess the possible mechanistic roles played by oxidative stress, increases in arginase activity and pro-inflammatory cytokines levels. For this purpose, 38, 13-month-old C57BL/6 male mice were utilized. Control animals (n=13) were fed a regular chow for a period of 10 months. T2D (n=25) animals were provided a single injection of streptozotocin (75 mg/kg/IP) and fed a 60% high fat diet (HFD) for the same period of time. To track changes in cardiac structure and function serial echocardiography was performed. At the end of the study, blood samples were collected via a cardiac puncture and plasma samples frozen. Hearts were sectioned and allocated for either rapid freezing (biochemical studies) or fixed in formalin for histological analysis. Biochemical assays using blood and/or tissue samples included determinations for arginase activity, pro-inflammatory cytokines, protein carbonylation, glucose tolerance tests, Western blots and plasma metabolomics. Glucose tolerance tests confirmed the development of T2D in the HFD animal group. Serial echo yielded moderate increases in cardiac mass with aging that were greater in T2D animals. Fractional shortening (figure) significantly decreased by 16 months of age in T2D animals yielding a value of 28% at 23 months vs. 40% in aged controls. Ejection fraction also significantly decreased by 16 months of age in T2D animals yielding 55% at 23 months vs. aged animals (70%). Western blots of myocardium samples documented increases in fibronectin, type III collagen and smooth muscle actin with T2D which were confirmed by immunohistochemistry. Significant increases in plasma protein carbonyls, TGF-β1, TNF-α, IL1β and IFN-γ occurred with aging and further increased with T2D. Untargeted metabolomics and cheminformatics revealed clear differences in the plasma metabolome of the HFD mice vs. controls. Multiple lipid metabolites linked to insulin resistance were dysregulated, including C16:0 and C18:0 acyl carnitines (up-modulated) and saturated and unsaturated phosphocholine-containing lipids (up and down-modulated). In summary, we document the development of cardiac hypertrophy with aging that is aggravated by T2D. Systolic dysfunction occurred as a late age associated event with T2D. Increases in OS markers and pro-inflammatory cytokines occurred with aging and were aggravated with T2D. These changes were accompanied by increases in myocardial and blood arginase activity and TGF-β1 levels which may account for the development of fibrosis and may be associated with altered plasma metabolomics.
  52. FASEB J. 2022 May;36 Suppl 1
      BACKGROUND: Aging is a risk factor for cardiovascular diseases, which are major causes of disability and mortality in the elderly. Endothelial dysfunction and an imbalanced gut microbial ecology play a pivotal role in aging-associated vascular complications. Dietary change may be an effective strategy to improve vascular health. In this study, we tested the hypothesis that dietary blueberries ameliorate vascular complications and gut dysbiosis in aged mice.METHODS: Aged mice (17-month-old C57BL/6J male, Jackson Laboratory) were fed a control diet (O) or freeze-dried wild blueberry powder supplemented diet (3.8% in diet) (OB) for 15 weeks. Young mice (2-month-old) consumed a control diet (Y) or blueberry supplemented diet (YB) for an identical duration. Based on normalization to body surface area, the blueberry dose is equivalent to 1.5 human servings of blueberry (~240 g) per day. Metabolic parameters, vascular function and vascular inflammation were assessed at the end of the treatment period. Vascular inflammation was assessed by measuring the binding of fluorescent labelled mouse monocytic WEHI78/24 cells to the vascular endothelium. Mesenteric arteries were used to assess vascular function using isometric procedures. Microbial profiling was done using 16s rRNA amplification.
    RESULTS: Metabolic parameters such as body weight, food intake and blood glucose were similar among the groups. Old mice (O) exhibited improved glucose tolerance compared to young mice (Y). Blueberry supplementation did not alter glucose tolerance in young (YB) or old mice (OB). O vs Y had increased monocyte binding to vascular endothelium indicating enhanced vascular inflammation, but this was reduced by blueberry supplementation in OB vs O. Endothelium-dependent vasorelaxation to acetylcholine and endothelium-independent vasorelaxation to sodium nitroprusside were similar among the groups. Microbial profiling indicated changes in the composition of gut microbiome among the groups. α-diversity indices such as Chao and observed species were similar at the phylum level but were different at the genus level among groups. β-diversity, which represents compositional differences among groups, was different at the phylum and genus levels. Further, the relative abundance of gut microbes at different taxa levels were altered between O vs Y and OB vs O mice. Importantly, the relative abundance of genera Candidatus Saccharimonas and Enterorhabdus were decreased whereas Muribaculum was increased in O vs Y mice. Blueberry supplementation improved the relative abundance of these three genera in OB vs O mice.
    CONCLUSION: Blueberry supplementation improves aging induced vascular inflammation in C57BL/6J mice without altering the metabolic milieu indicating the direct effect of blueberries on vasculature. Our study also provides evidence for changes in the composition of gut microbiome which might mediate some of the effects of blueberry supplementation in aged mice.
  53. Aging Cell. 2022 May 12. e13608
      DNA methylation (DNAm) has been reported to be associated with many diseases and with mortality. We hypothesized that the integration of DNAm with clinical risk factors would improve mortality prediction. We performed an epigenome-wide association study of whole blood DNAm in relation to mortality in 15 cohorts (n = 15,013). During a mean follow-up of 10 years, there were 4314 deaths from all causes including 1235 cardiovascular disease (CVD) deaths and 868 cancer deaths. Ancestry-stratified meta-analysis of all-cause mortality identified 163 CpGs in European ancestry (EA) and 17 in African ancestry (AA) participants at p < 1 × 10-7 , of which 41 (EA) and 16 (AA) were also associated with CVD death, and 15 (EA) and 9 (AA) with cancer death. We built DNAm-based prediction models for all-cause mortality that predicted mortality risk after adjusting for clinical risk factors. The mortality prediction model trained by integrating DNAm with clinical risk factors showed an improvement in prediction of cancer death with 5% increase in the C-index in a replication cohort, compared with the model including clinical risk factors alone. Mendelian randomization identified 15 putatively causal CpGs in relation to longevity, CVD, or cancer risk. For example, cg06885782 (in KCNQ4) was positively associated with risk for prostate cancer (Beta = 1.2, PMR  = 4.1 × 10-4 ) and negatively associated with longevity (Beta = -1.9, PMR  = 0.02). Pathway analysis revealed that genes associated with mortality-related CpGs are enriched for immune- and cancer-related pathways. We identified replicable DNAm signatures of mortality and demonstrated the potential utility of CpGs as informative biomarkers for prediction of mortality risk.
    Keywords:  DNA methylation; cancer; cardiovascular disease; machine learning; mortality
  54. Nat Metab. 2022 May 09.
      The severity and mortality of COVID-19 are associated with pre-existing medical comorbidities such as diabetes mellitus. However, the underlying causes for increased susceptibility to viral infection in patients with diabetes is not fully understood. Here we identify several small-molecule metabolites from human blood with effective antiviral activity against SARS-CoV-2, one of which, 1,5-anhydro-D-glucitol (1,5-AG), is associated with diabetes mellitus. The serum 1,5-AG level is significantly lower in patients with diabetes. In vitro, the level of SARS-CoV-2 replication is higher in the presence of serum from patients with diabetes than from healthy individuals and this is counteracted by supplementation of 1,5-AG to the serum from patients. Diabetic (db/db) mice undergo SARS-CoV-2 infection accompanied by much higher viral loads and more severe respiratory tissue damage when compared to wild-type mice. Sustained supplementation of 1,5-AG in diabetic mice reduces SARS-CoV-2 loads and disease severity to similar levels in nondiabetic mice. Mechanistically, 1,5-AG directly binds the S2 subunit of the SARS-CoV-2 spike protein, thereby interrupting spike-mediated virus-host membrane fusion. Our results reveal a mechanism that contributes to COVID-19 pathogenesis in the diabetic population and suggest that 1,5-AG supplementation may be beneficial to diabetic patients against severe COVID-19.