Kallistatin attenuates endothelial senescence by modulating Let‐7g‐mediated miR‐34a‐SIRT1‐eNOS pathway

Kallistatin, a plasma protein, protects against vascular and organ injury. This study is aimed to investigate the role and mechanism of kallistatin in endothelial senescence. Kallistatin inhibited H₂O₂‐induced senescence in human endothelial cells, as indicated by reduced senescence‐associated‐β‐galactosidase activity, p16^INK4a and plasminogen activator inhibitor‐1 expression, and elevated telomerase activity. Kallistatin blocked H₂O₂‐induced superoxide formation, NADPH oxidase levels and VCAM‐1, ICAM‐1, IL‐6 and miR‐34a synthesis. Kallistatin reversed H₂O₂‐mediated inhibition of endothelial nitric oxide synthase (eNOS), SIRT1, catalase and superoxide dismutase (SOD)‐2 expression, and kallistatin alone stimulated the synthesis of these antioxidant enzymes. Moreover, kallistatin's anti‐senescence and anti‐oxidant effects were attributed to SIRT1‐mediated eNOS pathway. Kallistatin, via interaction with tyrosine kinase, up‐regulated Let‐7g, whereas Let‐7g inhibitor abolished kallistatin's effects on miR‐34a and SIRT1/eNOS synthesis, leading to inhibition of senescence, oxidative stress and inflammation. Furthermore, lung endothelial cells isolated from endothelium‐specific kallistatin knockout mice displayed marked reduction in mouse kallistatin levels. Kallistatin deficiency in mouse endothelial cells exacerbated senescence, oxidative stress and inflammation compared to wild‐type mouse endothelial cells, and H₂O₂ treatment further magnified these effects. Kallistatin deficiency caused marked reduction in Let‐7g, SIRT1, eNOS, catalase and SOD‐1 mRNA levels, and elevated miR‐34a synthesis in mouse endothelial cells. These findings indicate that endogenous kallistatin through novel mechanisms protects against endothelial senescence by modulating Let‐7g‐mediated miR‐34a‐SIRT1‐eNOS pathway.     

The plant defensin RsAFP2 induces cell wall stress, septin mislocalization and accumulation of ceramides in Candida albicans

The antifungal plant defensin RsAFP2 isolated from radish interacts with fungal glucosylceramides and induces apoptosis in Candida albicans. To further unravel the mechanism of RsAFP2 antifungal action and tolerance mechanisms, we screened a library of 2868 heterozygous C. albicans deletion mutants and identified 30 RsAFP2‐hypersensitive mutants. The most prominent group of RsAFP2 tolerance genes was involved in cell wall integrity and hyphal growth/septin ring formation. Consistent with these genetic data, we demonstrated that RsAFP2 interacts with the cell wall of C. albicans, which also contains glucosylceramides, and activates the cell wall integrity pathway. Moreover, we found that RsAFP2 induces mislocalization of septins and blocks the yeast‐to‐hypha transition in C. albicans. Increased ceramide levels have previously been shown to result in apoptosis and septin mislocalization. Therefore, ceramide levels in C. albicans membranes were analysed following RsAFP2 treatment and, as expected, increased accumulation of phytoC24‐ceramides in membranes of RsAFP2‐treated C. albicans cells was detected. This is the first report on the interaction of a plant defensin with glucosylceramides in the fungal cell wall, causing cell wall stress, and on the effects of a defensin on septin localization and ceramide accumulation.     

Phospholipase D modulation by ceramide in senescence

Phospholipase D (PLD) has been implicated in mitotic regulation and has been shown to be defective in cells following replicative senescence. We examined the source of changes in PLD activity in senescent human umbilical vein endothelial cells and in human diploid fibroblasts. Using fractionated cell components we found that the cytosolic components were necessary for maximum PLD activation. In comparison to low-passage cells, senescent cells showed a profound lack of PLD activatability. By recombining fractionated components from senescent and low-passage cells, we found that in senescence the membrane component is defective in activating PLD implicating either the PLD enzyme itself or its interaction with PKC and/or ARF. The sphingolipid ceramide has been implicated in mediating senescence. Treatment with ceramide resulted in a decrease in PLD activity, implicating ceramide as the mediator of the inhibition.     

S‐sulfhydration of MEK1 leads to PARP‐1 activation and DNA damage repair

The repair of DNA damage is fundamental to normal cell development and replication. Hydrogen sulfide (H₂S) is a novel gasotransmitter that has been reported to protect cellular aging. Here, we show that H₂S attenuates DNA damage in human endothelial cells and fibroblasts by S‐sulfhydrating MEK1 at cysteine 341, which leads to PARP‐1 activation. H₂S‐induced MEK1 S‐sulfhydration facilitates the translocation of phosphorylated ERK1/2 into nucleus, where it activates PARP‐1 through direct interaction. Mutation of MEK1 cysteine 341 inhibits ERK phosphorylation and PARP‐1 activation. In the presence of H₂S, activated PARP‐1 recruits XRCC1 and DNA ligase III to DNA breaks to mediate DNA damage repair, and cells are protected from senescence.     

Phenyl 2‐pyridyl ketoxime induces cellular senescence‐like alterations via nitric oxide production in human diploid fibroblasts

Phenyl‐2‐pyridyl ketoxime (PPKO) was found to be one of the small molecules enriched in the extracellular matrix of near‐senescent human diploid fibroblasts (HDFs). Treatment of young HDFs with PPKO reduced the viability of young HDFs in a dose‐ and time‐dependent manner and resulted in senescence‐associated β‐galactosidase (SA‐β‐gal) staining and G2/M cell cycle arrest. In addition, the levels of some senescence‐associated proteins, such as phosphorylated ERK1/2, caveolin‐1, p53, p16^ink4a, and p21^waf1, were elevated in PPKO‐treated cells. To monitor the effect of PPKO on cell stress responses, reactive oxygen species (ROS) production was examined by flow cytometry. After PPKO treatment, ROS levels transiently increased at 30 min but then returned to baseline at 60 min. The levels of some antioxidant enzymes, such as catalase, peroxiredoxin II and glutathione peroxidase I, were transiently induced by PPKO treatment. SOD II levels increased gradually, whereas the SOD I and III levels were biphasic during the experimental periods after PPKO treatment. Cellular senescence induced by PPKO was suppressed by chemical antioxidants, such as N‐acetylcysteine, 2,2,6,6‐tetramethylpiperidinyloxy, and L‐buthionine‐(S,R)‐sulfoximine. Furthermore, PPKO increased nitric oxide (NO) production via inducible NO synthase (iNOS) in HDFs. In the presence of NOS inhibitors, such as L‐NG‐nitroarginine methyl ester and L‐NG‐monomethylarginine, PPKO‐induced transient NO production and SA‐β‐gal staining were abrogated. Taken together, these results suggest that PPKO induces cellular senescence in association with transient ROS and NO production and the subsequent induction of senescence‐associated proteins .     

Rapamycin inhibits the secretory phenotype of senescent cells by a Nrf2‐independent mechanism

Senescent cells contribute to age‐related pathology and loss of function, and their selective removal improves physiological function and extends longevity. Rapamycin, an inhibitor of mTOR, inhibits cell senescence in vitro and increases longevity in several species. Nrf2 levels have been shown to decrease with aging and silencing Nrf2 gene induces premature senescence. Therefore, we explored whether Nrf2 is involved in the mechanism by which rapamycin delays cell senescence. In wild‐type (WT) mouse fibroblasts, rapamycin increased the levels of Nrf2, and this correlates with the activation of autophagy and a reduction in the induction of cell senescence, as measured by SA‐β‐galactosidase (β‐gal) staining, senescence‐associated secretory phenotype (SASP), and p16 and p21 molecular markers. In Nrf2KO fibroblasts, however, rapamycin still decreased β‐gal staining and the SASP, but rapamycin did not activate the autophagy pathway or decrease p16 and p21 levels. These observations were further confirmed in vivo using Nrf2KO mice, where rapamycin treatment led to a decrease in β‐gal staining and pro‐inflammatory cytokines in serum and fat tissue; however, p16 levels were not significantly decreased in fat tissue. Consistent with literature demonstrating that the Stat3 pathway is linked to the production of SASP, we found that rapamycin decreased activation of the Stat3 pathway in cells or tissue samples from both WT and Nrf2KO mice. Our data thus suggest that cell senescence is a complex process that involves at least two arms, and rapamycin uses Nrf2 to regulate cell cycle arrest, but not the production of SASP.     

Endothelial cell telomere dysfunction induces senescence and results in vascular and metabolic impairments

In advanced age, increases in oxidative stress and inflammation impair endothelial function, which contributes to the development of cardiovascular disease (CVD). One plausible source of this oxidative stress and inflammation is an increase in the abundance of senescent endothelial cells. Cellular senescence is a cell cycle arrest that occurs in response to various damaging stimuli. In the present study, we tested the hypothesis that advanced age results in endothelial cell telomere dysfunction that induces senescence. In both human and mouse endothelial cells, advanced age resulted in an increased abundance of dysfunctional telomeres, characterized by activation of DNA damage signaling at telomeric DNA. To test whether this results in senescence, we selectively reduced the telomere shelterin protein telomere repeat binding factor 2 (Trf2) from endothelial cells of young mice. Trf2 reduction increased endothelial cell telomere dysfunction and resulted in cellular senescence. Furthermore, induction of endothelial cell telomere dysfunction increased inflammatory signaling and oxidative stress, resulting in impairments in endothelial function. Finally, we demonstrate that endothelial cell telomere dysfunction-induced senescence impairs glucose tolerance. This likely occurs through increases in inflammatory signaling in the liver and adipose tissue, as well as reductions in microvascular density and vasodilation to metabolic stimuli. Cumulatively, the findings of the present study identify age-related telomere dysfunction as a mechanism that leads to endothelial cell senescence. Furthermore, these data provide compelling evidence that senescent endothelial cells contribute to age-related increases in oxidative stress and inflammation that impair arterial and metabolic function.     

Acheron regulates vascular endothelial proliferation and angiogenesis together with Id1 during wound healing

RNA binding protein acheron has proved to be either the mediator of integrin‐extracellular matrix interactions or the regulatory factor that participates in vertebrate development, cell differentiation and cell death. We report the role of acheron in vascular endothelial proliferation, angiogenesis and wound healing post‐trauma. Co‐immunoprecipitation showed that Acheron forms a ternary complex with β1 integrin and Id1 in human umbilical vein endothelial cells following stimulation with serious trauma serum. Acheron, vascular endothelial growth factor (VEGF), and β1 integrin mRNA expression was apparently inhibited, and capillary density and wound healing rate also were reduced in Id1‐deficient mice trauma model. Acheron together with Id1 significantly induces VEGF, not CD105 level inhibition by serious trauma serum for 24 h. In conclusion, we have demonstrated that acheron may be an effective mediator of promoting endothelial proliferation, angiogenesis and wound healing probably by regulating VEGF together with Id1. Copyright © 2011 John Wiley & Sons, Ltd.     

Ceramide plays a prominent role in MDA‐7/IL‐24‐induced cancer‐specific apoptosis

Melanoma differentiation associated gene‐7/interleukin‐24 (mda‐7/IL‐24) uniquely displays broad cancer‐specific apoptosis‐inducing activity through induction of endoplasmic reticulum (ER) stress. We hypothesize that ceramide, a promoter of apoptosis, might contribute to mda‐7/IL‐24 induction of apoptosis. Ad.mda‐7‐infected tumor cells, but not normal cells, showed increased ceramide accumulation. Infection with Ad.mda‐7 induced a marked increase in various ceramides (C16, C24, C24:1) selectively in prostate cancer cells. Inhibiting the enzyme serine palmitoyltransferase (SPT) using the potent SPT inhibitor myriocin (ISP1), impaired mda‐7/IL‐24‐induced apoptosis and ceramide production, suggesting that ceramide formation caused by Ad.mda‐7 occurs through de novo synthesis of ceramide and that ceramide is required for mda‐7/IL‐24‐induced cell death. Fumonisin B1 (FB1) elevated ceramide formation as well as apoptosis induced by Ad.mda‐7, suggesting that ceramide formation may also occur through the salvage pathway. Additionally, Ad.mda‐7 infection enhanced expression of acid sphingomyelinase (ASMase) with a concomitant increase in ASMase activity and decreased sphingomyelin in cancer cells. ASMase silencing by RNA interference inhibited the decreased cell viability and ceramide formation after Ad.mda‐7 infection. Ad.mda‐7 activated protein phosphatase 2A (PP2A) and promoted dephosphorylation of the anti‐apoptotic molecule BCL‐2, a downstream ceramide‐mediated pathway of mda‐7/IL‐24 action. Pretreatment of cells with FB1 or ISP‐1 abolished the induction of ER stress markers (BiP/GRP78, GADD153 and pospho‐eIF2α) triggered by Ad.mda‐7 infection indicating that ceramide mediates ER stress induction by Ad.mda‐7. Additionally, recombinant MDA‐7/IL‐24 protein induced cancer‐specific production of ceramide. These studies define ceramide as a key mediator of an ER stress pathway that may underlie mda‐7/IL‐24 induction of cancer‐specific killing. J. Cell. Physiol. 222: 546–555, 2010. © 2009 Wiley‐Liss, Inc.     

Angiotensin II‐induced redox‐sensitive SGLT1 and 2 expression promotes high glucose‐induced endothelial cell senescence

High glucose (HG)‐induced endothelial senescence and dysfunction contribute to the increased cardiovascular risk in diabetes. Empagliflozin, a selective sodium glucose co‐transporter2 (SGLT2) inhibitor, reduced the risk of cardiovascular mortality in type 2 diabetic patients but the protective mechanism remains unclear. This study examines the role of SGLT2 in HG‐induced endothelial senescence and dysfunction. Porcine coronary artery cultured endothelial cells (ECs) or segments were exposed to HG (25 mmol/L) before determination of senescence‐associated beta‐galactosidase activity, protein level by Western blot and immunofluorescence staining, mRNA by RT‐PCR, nitric oxide (NO) by electron paramagnetic resonance, oxidative stress using dihydroethidium and glucose uptake using 2‐NBD‐glucose. HG increased ECs senescence markers and oxidative stress, down‐regulated eNOS expression and NO formation, and induced the expression of VCAM‐1, tissue factor, and the local angiotensin system, all these effects were prevented by empagliflozin. Empagliflozin and LX‐4211 (dual SGLT1/2 inhibitor) reduced glucose uptake stimulated by HG and H₂O₂ in ECs. HG increased SGLT1 and 2 protein levels in cultured ECs and native endothelium. Inhibition of the angiotensin system prevented HG‐induced ECs senescence and SGLT1 and 2 expression. Thus, HG‐induced ECs ageing is driven by the local angiotensin system via the redox‐sensitive up‐regulation of SGLT1 and 2, and, in turn, enhanced glucotoxicity.     

Induced pluripotent stem cell‐conditional medium inhibits H9C2 cardiomyocytes apoptosis via autophagy flux and Wnt/β‐catenin pathway

Induced pluripotent stem cell‐derived conditioned medium (iPS‐CM) could improve cell viability in many types of cells and may be a better alternative for the treatment of myocardial infarction. This study aimed to examine the influence of iPS‐CM on anti‐apoptosis and the proliferation of H9C2 cardiomyocytes and investigate the underlying mechanisms. H9C2 cardiomyocytes were exposed to 200 μmol/L hydrogen peroxide (H₂O₂) for 24 hours with or without pre‐treatment with iPS‐CM. The ratio of apoptotic cells, the loss of mitochondrial membrane potential (△Ψm) and the levels of intracellular reactive oxygen species were analysed by flow cytometric analysis. The expression levels of BCL‐2 and BAX proteins were analysed by Western blot. Cell proliferation was assessed using cell cycle and EdU staining assays. To study cell senescence, senescence‐associated β‐galactosidase (SA‐β‐gal) staining was conducted. The levels of malondialdehyde, superoxide dismutase and glutathione were also quantified using commercially available enzymatic kits. The results showed that iPS‐CM containing basic fibroblast growth factor significantly reduced H₂O₂‐induced H9C2 cardiomyocyte apoptosis by activating the autophagy flux pathway, promoted cardiomyocyte proliferation by up‐regulating the Wnt/β‐catenin pathway and inhibited oxidative stress and cell senescence. In conclusion, iPS‐CM effectively enhanced the cell viability of H9C2 cardiomyocytes and could potentially be used to inhibit cardiomyocytes apoptosis to treat myocardial infarction in the future.     

Hydrogen sulfide reduces cell adhesion and relevant inflammatory triggering by preventing ADAM17‐dependent TNF‐α activation

H₂S is the third endogenous gaseous mediator, after nitric oxide and carbon monoxide, possessing pleiotropic effects, including cytoprotection and anti‐inflammatory action. We analyzed, in an in vitro model entailing monocyte adhesion to an endothelial monolayer, the changes induced by H₂S on various potential targets, including cytokines, chemokines, and proteases, playing a crucial role in inflammation and cell adhesion. Results show that H₂S prevents the increase in monocyte adhesion induced by tumor necrosis factor‐α (TNF‐α). Under these conditions, downregulation of monocyte chemoattractant protein‐1 (MCP‐1), chemokine C‐C motif receptor 2, and increase of cluster of differentiation 36 could be detected in monocytes. In endothelial cells, H₂S treatment reduces the increase in MCP‐1, inter‐cellular adhesion molecule‐1, vascular cell adhesion molecule‐1, and of a disintegrin and metalloproteinase metallopeptidase domain 17 (ADAM17), both at the gene expression and protein levels. Cystathionine γ‐lyase and 3‐mercaptopyruvate sulfurtransferase, the major H₂S forming enzymes, are downregulated in endothelial cells. In addition, H₂S significantly reduces activation of ADAM17 by PMA in endothelial cells, with consequent reduction of both ADAM17‐dependent TNF‐α ectodomain shedding and MCP‐1 release. In conclusion, H₂S is able to prevent endothelial activation by hampering endothelial activation, triggered by TNF‐α. The mechanism of this protective effect is mainly mediated by down‐modulation of ADAM17‐dependent TNF‐converting enzyme (TACE) activity with consequent inhibition of soluble TNF‐α shedding and its relevant MCP‐1 release in the medium. These results are discussed in the light of the potential protective role of H₂S in pro‐inflammatory and pro‐atherogenic processes, such as chronic renal failure. J. Cell. Biochem. 114: 1536–1548, 2013. © 2013 Wiley Periodicals, Inc.     

Senescence‐associated DNA methylation is stochastically acquired in subpopulations of mesenchymal stem cells

Replicative senescence has a major impact on function and integrity of cell preparations. This process is reflected by continuous DNA methylation (DNAm) changes at specific CpG dinucleotides in the course of in vitro culture, and such modifications can be used to estimate the state of cellular senescence for quality control of cell preparations. Still, it is unclear how senescence‐associated DNAm changes are regulated and whether they occur simultaneously across a cell population. In this study, we analyzed global DNAm profiles of human mesenchymal stem cells (MSCs) and human umbilical vein endothelial cells (HUVECs) to demonstrate that senescence‐associated DNAm changes are overall similar in these different cell types. Subsequently, an Epigenetic‐Senescence‐Signature, based on six CpGs, was either analyzed by pyrosequencing or by bar‐coded bisulfite amplicon sequencing. There was a good correlation between predicted and real passage numbers in bulk populations of MSCs (R² = 0.67) and HUVECs (R² = 0.97). However, when we analyzed the Epigenetic‐Senescence‐Signature in subclones of MSCs, the predictions revealed high variation and they were not related to the adipogenic or osteogenic differentiation potential of the subclones. Notably, in clonally derived subpopulations, the DNAm levels of neighboring CpGs differed extensively, indicating that these genomic regions are not synchronously modified during senescence. Taken together, senescence‐associated DNAm changes occur in a highly reproducible manner, but they are not synchronously co‐regulated. They rather appear to be acquired stochastically—potentially evoked by other epigenetic modifications.     

Terrein reduces age‐related inflammation induced by oxidative stress through Nrf2/ERK1/2/HO‐1 signalling in aged HDF cells

This study investigated whether multiple bioactivity of terrein such as anti‐inflammatory and anti‐oxidant inhibits age‐related inflammation by promoting an antioxidant response in aged human diploid fibroblast (HDF) cells. HDF cells were cultured serially for in vitro replicative senescence. To create the ageing cell phenotype, intermediate stage (PD31) HDF cells were brought to stress‐induced premature senescence (SIPS) using hydrogen peroxide (H₂O₂). Terrein increased cell viability even with H₂O₂ stress and reduced inflammatory molecules such as intracellular adhesion molecule‐1 (ICAM‐1), cyclooxygenase‐2 (COX‐2), interleukin‐1beta (IL‐1β) and tumour necrosis factor‐alpha (TNF‐α). Terrein reduced also phospho‐extracellular kinase receptor1/2 (p‐EKR1/2) signalling in aged HDF cells. SIPS cells were attenuated for age‐related biological markers including reactive oxygen species (ROS), senescence associated beta‐galactosidase (SA β‐gal.) and the aforementioned inflammatory molecules. Terrein induced the induction of anti‐oxidant molecules, copper/zinc‐superoxide defence (Cu/ZnSOD), manganese superoxide dismutase (MnSOD) and heme oxygenase‐1 (HO‐1) in SIPS cells. Terrein also alleviated reactive oxygen species formation through the Nrf2/HO‐1/p‐ERK1/2 pathway in aged cells. The results indicate that terrein has an alleviative function of age‐related inflammation characterized as an anti‐oxidant. Terrein might be a useful nutraceutical compound for anti‐ageing. Copyright © 2015 John Wiley & Sons, Ltd.