Modifications of proteoglycans produced by human skin fibroblast cultures during replicative senescence

The properties of proteoglycans (PGs) produced by normal human skin fibroblast were investigated with increasing passage. The increase of subculture number was associated with a constant increase in PG molecular size, which was particularly evident in cell layer extracts. In the cell layer, the ratio of DS-PGs/HS-PGs was markedly higher in early passage cultures. Moreover, the cell layer from young cells contained lower amounts of radioactivity incorporated into the most hydrophobic PG populations, suggesting that the PG core protein might also undergo significant modification with increasing subcultures. There was no significant difference in energy charge value between early and late passage cultures, whereas the NAD/NADH ratio was found to decrease markedly in senescent cells.     

A potential role for NEDD1 and the centrosome in senescence of mouse embryonic fibroblasts

Mouse embryonic fibroblasts (MEFs) are commonly grown in cell culture and are known to enter senescence after a low number of passages as a result of oxidative stress. Oxidative stress has also been suggested to promote centrosome disruption; however, the contribution of this organelle to senescence is poorly understood. Therefore, this study aimed to assess the role of the centrosome in oxidative stress induced-senescence using MEFs as a model. We demonstrate here that coincident with the entry of late-passage MEFs into senescence, there was an increase in supernumerary centrosomes, most likely due to centrosome fragmentation. In addition, disrupting the centrosome in early-passage MEFs by depletion of neural precursor cell expressed developmentally downregulated gene 1 (NEDD1) also resulted in centrosomal fragmentation and subsequent premature entry into senescence. These data show that a loss of centrosomal integrity may contribute to the entry of MEFs into senescence in culture, and that centrosomal disruption can cause senescence.     

Snake venoms promote stress-induced senescence in human fibroblasts

Snake venoms are widely studied in terms of their systemic toxicity and proteolytic, hemotoxic, neurotoxic, and cytotoxic activities. However, little is known about snake-venom-mediated effects when used at low, noncytotoxic concentrations. In the current study, two human fibroblast cell lines of different origin, namely WI-38 fetal lung fibroblasts and BJ foreskin fibroblasts were used to investigate snake-venom-induced adaptive response at a relatively noncytotoxic concentration (0.01 µg/ml). The venoms of Indochinese spitting cobra ( Naja siamensis), western green mamba ( Dendroaspis viridis), forest cobra ( Naja melanoleuca), and southern copperhead ( Agkistrodon contortrix) were considered. Snake venoms promoted FOXO3a-mediated oxidative stress response and to a lesser extent DNA damage response, which lead to changes in cell cycle regulators both at messenger RNA and protein levels, limited cell proliferation and migration, and induced cellular senescence. Taken together, we have shown for the first time that selected snake venoms may also exert adverse effects when used at relatively noncytotoxic concentrations.     

Attenuation of acute and chronic inflammation using compounds derived from plants

The appearance of excessive inflammatory activity is associated with onset of many disease states. Such non-productive responses are often the basis of the mortality consequent to incurring numerous disorders. The current outbreak of coronavirus disease 2019 caused by the virus “severe acute respiratory syndrome coronavirus 2” is a striking reflection of the inadequacy of current medical science to adequately address this issue. The usefulness of a range of materials of botanical origin in the attenuation of both chronic and acute inflammatory responses to various disease stressors is described. The properties of preparations of plant-based origin often parallel those of synthesized pharmacologics, but differ from them in some key respects. These differences can lead to more traditional preparations having distinct therapeutic advantages but also a number of specific shortcomings. The strengths and weaknesses of these materials are objectively contrasted with that of a more orthodox pharmacological approach. Each of these emphases in style has specific advantages and they should not be considered as competitors, but rather as accomplices in combating adverse states involving derangement of immune function.     

Cyclic Stretch Induces Inflammatory Cytokines via the Oxidative Stress and NF-ΚB Pathways Activation in Human Keratoconic Fibroblasts

The cornea is a load-bearing tissue. Lower biomechanical properties in the local tissue of keratoconic cornea evoke mechanical stress increase. Inflammatory cytokines have been shown to be over-expressed in patients with keratoconus. However, how mechanical stimuli are involved in the production of inflammatory cytokines in keratoconus remains unclear. The objective of the study is to determine the role of mechanical stretch in the regulation of inflammatory cytokines and the underlying mechanisms in keratoconus. Human keratoconic fibroblasts (hKCFs) were subjected to 12% cyclic mechanical stretch at 0.1 Hz or in static conditions as controls. N-acetyl cysteine (NAC) and pyrrolidine dithiocarbamate and pyrrolidine dithiocarbamate (PDTC) were used to inhibit reactive oxygen species (ROS) production and NF-κB pathway respectively. ROS production was measured using 2’,7’-dichlorodihydrofluorescindiacetate probe. Conditioned media and cell lysates were collected for protein assessment. Cyclic stretch-induced a higher production of intercellular cell adhesion molecule-1 (ICAM-1), tumor necrosis factor α (TNF-α), interleukin (IL)-6, and IL-8 in hKCFs than static controls. ROS was also elevated in response to cyclic stretch. Inhibition of ROS or NF-κB attenuated stretch-induced ICAM-1, TNF-α, IL-6, and IL-8. Inhibition of stretch-induced ROS production by NAC also attenuated NF-κB activation. Our findings suggest that mechanical stretch may induce the release of inflammatory cytokines by activating oxidative stress and NF-kB pathway, and ROS may positively control NF-κB signaling. Over-expression of inflammatory cytokines induced by mechanical stretch may play a role in progression of keratoconus.     

Management of multicellular senescence and oxidative stress

Progressively sophisticated understanding of cellular and molecular processes that contribute to age‐related physical deterioration is being gained from ongoing research into cancer, chronic inflammatory syndromes and other serious disorders that increase with age. Particularly valuable insight has resulted from characterization of how senescent cells affect the tissues in which they form in ways that decrease an organism's overall viability. Increasingly, the underlying pathophysiology of ageing is recognized as a consequence of oxidative damage. This leads to hyperactivity of cell growth pathways, prominently including mTOR (mammalian target of rapamycin), that contribute to a build‐up in cells of toxic aggregates such as progerin (a mutant nuclear cytoskeletal protein), lipofuscin and other cellular debris, triggering formation of senescent cellular phenotypes, which interact destructively with surrounding tissue. Indeed, senescent cell ablation dramatically inhibits physical deterioration in progeroid (age‐accelerated) mice. This review explores ways in which oxidative stress creates ageing‐associated cellular damage and triggers induction of the cell death/survival programs’ apoptosis, necrosis, autophagy and ‘necroapoptophagy’. The concept of ‘necroapoptophagy’ is presented here as a strategy for varying tissue oxidative stress intensity in ways that induce differential activation of death versus survival programs, resulting in enhanced and sustained representation of healthy functional cells. These strategies are discussed in the context of specialized mesenchymal stromal cells with the potential to synergize with telocytes in stabilizing engrafted progenitor cells, thereby extending periods of healthy life. Information and concepts are summarized in a hypothetical approach to suppressing whole‐organism senescence, with methods drawn from emerging understandings of ageing, gained from Cnidarians (jellyfish, corals and anemones) that undergo a unique form of cellular regeneration, potentially conferring open‐ended lifespans.     

Inhibition of thioredoxin reductase 1 by caveolin 1 promotes stress‐induced premature senescence

Thioredoxin reductase 1 (TrxR1) is an important antioxidant enzyme that controls cellular redox homeostasis. By using a proteomic‐based approach, here we identify TrxR1 as a caveolar membrane‐resident protein. We show that caveolin 1, the structural protein component of caveolae, is a TrxR1‐binding protein by demonstrating that the scaffolding domain of caveolin 1 (amino acids 82–101) binds directly to the caveolin‐binding motif (CBM) of TrxR1 (amino acids 454–463). We also show that overexpression of caveolin 1 inhibits TrxR activity, whereas a lack of caveolin 1 activates TrxR, both in vitro and in vivo. Expression of a peptide corresponding to the caveolin 1 scaffolding domain is sufficient to inhibit TrxR activity. A TrxR1 mutant lacking the CBM, which fails to localize to caveolae and bind to caveolin 1, is constitutively active and inhibits oxidative‐stress‐mediated activation of the p53/p21^Waf1/Cip1 pathway and induction of premature senescence. Finally, we show that caveolin 1 expression inhibits TrxR1‐mediated cell transformation. Thus, caveolin 1 links free radicals to activation of the p53/p21^Waf1/Cip1 pathway and induction of cellular senescence by acting as an endogenous inhibitor of TrxR1.     

Methionine restriction slows down senescence in human diploid fibroblasts

Methionine restriction (MetR) extends lifespan in animal models including rodents. Using human diploid fibroblasts (HDF), we report here that MetR significantly extends their replicative lifespan, thereby postponing cellular senescence. MetR significantly decreased activity of mitochondrial complex IV and diminished the accumulation of reactive oxygen species. Lifespan extension was accompanied by a significant decrease in the levels of subunits of mitochondrial complex IV, but also complex I, which was due to a decreased translation rate of several mtDNA‐encoded subunits. Together, these findings indicate that MetR slows down aging in human cells by modulating mitochondrial protein synthesis and respiratory chain assembly.     

Recent advances in cellular senescence, cancer and aging

How much do we know about the biology of aging from cell culture studies? Most normal somatic cells have a finite potential to divide due to a process termed cellular or replicative senescence. A growing body of evidence suggests that senescence evolved to protect higher eukaryotes, particularly mammals, from developing cancer. We now know that telomere shortening, due to the biochemistry of DNA replication, induces replicative senescence in human cells. However, in rodent cells, replicative senescence occurs despite very long telomeres. Recent findings suggest that replicative senescence is just the tip of the iceberg of a more general process termed cellular senescence. It appears that cellular senescence is a response to potentially oncogenic insults, including oxidative damage. In young organisms, growth arrest by cell senescence suppresses tumor development, but later in life, due to the accumulation of senescent cells which secret factors that can disrupt tissues during aging, cellular senescence promotes tumorigenesis. Therefore, antagonistic pleiotropy may explain in part, if not in whole, the apparently paradoxical effects of cellular senescence, though this still remains an open question.     

Glutathione produced by Rhizobium tropici is important to prevent early senescence in common bean nodules

In this paper, we examine the importance of glutathione in symbiosis, using a glutathione biosynthetic gshB mutant derived from Rhizobium tropici CIAT899, a common bean (Phaseolus vulgaris) endosymbiont. Plants infected with the mutant strain presented a delayed nodulation phenotype and a reduction in the dry weight of aerial part of plants, suggesting diminished nitrogen-fixation activity. In addition, bacterial gshB expression was assayed in wild-type infected nodules, during the different steps of nodulation, and found to increase in mature and early senescent nodules. Conspicuously, nodules induced by gshB mutant bacteria presented an early senescent pattern, which was associated with increased levels of superoxide accumulation. These results provide a direct evidence of the role of bacterial glutathione in protecting nodules from reactive oxygen species, which may determine nodule senescence.     

Metformin alleviates oxidative stress-induced senescence of human lens epithelial cells via AMPK activation and autophagic flux restoration

Cataracts are the leading cause of blindness worldwide owing to the increasing proportion of elderly individuals in the population. The purpose of this study was to investigate whether metformin could alleviate the occurrence and development of age-related cataract (ARC) and the underlying mechanism. In the present study, we established a senescence model induced by oxidative stress, which was confirmed by measuring β-galactosidase activity, qRT-PCR and Western blotting. In addition, we showed that metformin alleviated the oxidative stress-induced senescence of HLE-B3 cells via the activation of AMPK. Next, we provided evidence that oxidative stress impaired autophagic flux and induced lysosomal dysfunction. Subsequently, we found that metformin restored autophagic flux that had been impaired by oxidative stress by activating AMPK. Additionally, we found that metformin suppressed HLE-B3 cell senescence by improving lysosomal function and inactivating mTOR. Furthermore, the inactivation of AMPK, impairment of autophagic flux and lysosomal dysfunction were observed in the human lens epithelium of ARC. In summary, our data suggest that the activation of AMPK may be a potential strategy for preventing ARC, and metformin may be an emerging candidate to alleviate the formation and development of ARC.     

Disc cell senescence in intervertebral disc degeneration: Causes and molecular pathways

The accumulation of senescent disc cells in degenerative intervertebral disc (IVD) suggests the detrimental roles of cell senescence in the pathogenesis of intervertebral disc degeneration (IDD). Disc cell senescence decreased the number of functional cells in IVD. Moreover, the senescent disc cells were supposed to accelerate the process of IDD via their aberrant paracrine effects by which senescent cells cause the senescence of neighboring cells and enhance the matrix catabolism and inflammation in IVD. Thus, anti-senescence has been proposed as a novel therapeutic target for IDD. However, the development of anti-senescence therapy is based on our understanding of the molecular mechanism of disc cell senescence. In this review, we focused on the molecular mechanism of disc cell senescence, including the causes and various molecular pathways. We found that, during the process of IDD, age-related damages together with degenerative external stimuli activated both p53-p21-Rb and p16-Rb pathways to induce disc cell senescence. Meanwhile, disc cell senescence was regulated by multiple signaling pathways, suggesting the complex regulating network of disc cell senescence. To understand the mechanism of disc cell senescence better contributes to developing the anti-senescence-based therapies for IDD.     

Antioxidative protection in a drought-resistant maize strain during leaf senescence

Leaves of 7- and 18-day-old plants of two maize strains, one resistant (LIZA) and one sensitive (LG11) to water stress, were floated in 1 m M paraquat and 1 m M H₂O₂ for 12 h in light and in darkness. The aim of this work was to analyse the effects of these substances on the activities of enzymes involved in the scavenging of active oxygen species during senescence. Three senescence parameters; chlorophyll loss, lipid peroxidation and conductivity; showed a general cell damage caused by both oxidative treatments and revealed a higher tolerance of LIZA than LG11 to paraquat and H₂O₂ both in light and in darkness. Activities of antioxidative enzymes increased by the effect of oxidative treatments in young and senescent leaves of the drought-resistant maize strain LIZA. These increases were about 3-to 6-fold in glutathione reductase. 3-to 4-fold in superoxide dismutase and 2-fold in ascorbate peroxidase activities. The possible correlation between water stress resistance. senescence and the potential of antioxidant enzymes was analysed.     

Hyperosmolarity induced by high glucose promotes senescence in human glomerular mesangial cells

Hyperglycemia is involved in the diabetic complication of different organs and can elevate serum osmolarity. Here, we tested whether hyperosmolarity promoted by high glucose levels induces cellular senescence in renal cells. We treated Wistar rats with streptozotocin to induce diabetes or with consecutive daily injections of mannitol to increase serum osmolarity and analyzed p53 and p16 genes in renal cortex by immunohistochemistry. Both diabetic and mannitol treated rats showed a significant increase in serum osmolarity, without significant signs of renal dysfunction, but associated with increased staining for p53 and p16 in the renal cortex. An increase in p53 and p16 expression was also found in renal cortex slices and glomeruli isolated from healthy rats, which were later treated with 30 mM glucose or mannitol. Intracellular mechanisms involved were analyzed in cultured human glomerular mesangial cells treated with 30 mM glucose or mannitol. After treatments, cells showed increased p53, p21 and p16 expression and elevated senescence-associated β-galactosidase activity. Senescence was prevented when myo-inositol was added before treatment. High glucose or mannitol induced constitutive activation of Ras and ERK pathways which, in turn, were activated by oxidative stress. In summary, hyperosmolarity induced renal senescence, particularly in glomerular mesangial cells, increasing oxidative stress, which constitutively activated Ras-ERK 1/2 pathway. Cellular senescence could contribute to the organ dysfunction associated with diabetes.     

Bmal1-deficient mouse fibroblast cells do not provide premature cellular senescence in vitro

Bmal1 is a core circadian clock gene. Bmal1^?/? mice show disruption of the clock and premature aging phenotypes with a short lifespan. However, little is known whether disruption of Bmal1 leads to premature aging at cellular level. Here, we established primary mouse embryonic fibroblast (MEF) cells derived from Bmal1^?/? mice and investigated its effects on cellular senescence. Unexpectedly, Bmal1^?/? primary MEFs that showed disrupted circadian oscillation underwent neither premature replicative nor stress-induced cellular senescence. Our results therefore uncover that Bmal1 is not required for in vitro cellular senescence, suggesting that circadian clock does not control in vitro cellular senescence.     

Induction of premature senescence in cardiomyocytes by doxorubicin as a novel mechanism of myocardial damage

Cellular senescence is an important phenomenon in decreased cellular function. Recently, it was shown that cellular senescence is induced in proliferating cells within a short period of time by oxidative stresses. This phenomenon is known as premature senescence. However, it is still unknown whether premature senescence can be also induced in cardiomyocytes. The aim of the present study was to investigate whether a senescence-like phenotype can be induced in cardiomyocytes by oxidative stress. In cardiomyocytes obtained from aged rats (24 months of age), the staining for senescence-associated beta-galactosidase increased significantly and the protein or RNA levels of cyclin-dependent kinase inhibitors increased compared to those of young rats. Decreased cardiac troponin I phosphorylation and telomerase activity were also observed in aged cardiomyocytes. Treatment of cultured neonatal rat cardiomyocytes with a low concentration of doxorubicin (DOX) (10(-7) mol L(-1)) did not induce apoptosis but did induce oxidative stress, which was confirmed by 2',7'-dichlorofluorescin diacetate staining. In DOX-treated neonatal cardiomyocytes, increased positive staining for senescence-associated beta-galactosidase, cdk-I expression, decreased cardiac troponin I phosphorylation, and decreased telomerase activity were observed, as aged cardiomyocytes. Alterations in mRNA expression typically seen in aged cells were observed in DOX-treated neonatal cardiomyocytes. We also found that promyelocytic leukemia protein and acetylated p53, key proteins involved in stress-induced premature senescence in proliferating cells, were associated with cellular alterations of senescence in DOX-treated cardiomyocytes. In conclusion, cardiomyocytes treated with DOX showed characteristic changes similar to cardiomyocytes of aged rats. promyelocytic leukemia-related p53 acetylation may be an underlying mechanism of senescence-like alterations in cardiomyocytes. These findings indicate a novel mechanism of myocardial dysfunction induced by oxidative stress.     

Effect of water stress on some oxidative enzymes and senescence in Vigna seedlings

Seedlings of Vigna catjang Endl. were subjected to water stress for 6, S and 10 days by withholding water to investigate the activities of some oxidative enzymes and the pattern of senescence in leaves of 17-day-old seedlings undergoing water stress. Increasing duration of stress produced a proportional increase in the activities of IAA-oxidase, AA-oxidase, peroxidase and glycolate oxidase but decreased catalase activity and the contents of both chlorophyll and protein, hastening senescence. Leaf water potential and relative water content were also lowered with incresing duration of stress. Permeability was increased in leaf tissue undergoing water stress for 8 days. Seed treatment with CaCl₂ (10⁻² and 10⁻¹⁴ M ) for 6 h improved the water status of leaves, decreased tissue permeability, activities of oxidative enzymes, decline of chlorophyll and protein contents and delayed senescence compared to untreated water stressed plants.     

HIV protease inhibitors induce senescence and alter osteoblastic potential of human bone marrow mesenchymal stem cells: beneficial effect of pravastatin

HIV‐infected patients receiving antiretroviral therapy present an increased prevalence of age‐related comorbidities, including osteoporosis. HIV protease inhibitors (PIs) have been suspected to participate to bone loss, but the mechanisms involved are unknown. In endothelial cells, some PIs have been shown to induce the accumulation of farnesylated prelamin‐A, a biomarker of cell aging leading to cell senescence. Herein, we hypothesized that these PIs could induce premature aging of osteoblast precursors, human bone marrow mesenchymal stem cells (MSCs), and affect their capacity to differentiate into osteoblasts. Senescence was studied in proliferating human MSCs after a 30‐day exposure to atazanavir and lopinavir with or without ritonavir. When compared to untreated cells, PI‐treated MSCs had a reduced proliferative capacity that worsened with increasing passages. PI treatment led to increased oxidative stress and expression of senescence markers, including prelamin‐A. Pravastatin, which blocks prelamin‐A farnesylation, prevented PI‐induced senescence and oxidative stress, while treatment with antioxidants partly reversed these effects. Moreover, senescent MSCs presented a decreased osteoblastic potential, which was restored by pravastatin treatment. Because age‐related bone loss is associated with increased bone marrow fat, we also evaluated the capacity of PI‐treated MSCs to differentiate into adipocyte. We observed an altered adipocyte differentiation in PI‐treated MSCs that was reverted by pravastatin. We have shown that some PIs alter osteoblast formation by affecting their differentiation potential in association with altered senescence in MSCs, with a beneficial effect of statin. These data corroborate the clinical observations and allow new insight into pathophysiological mechanisms of PI‐induced bone loss in HIV‐infected patients.