Vascular smooth muscle cell senescence and age-related diseases: State of the art

Aging is a worldwide challenge, and it is accompanied by the accumulation of senescent cells. Cellular senescence is traditionally defined as permanent cell growth arrest and currently includes the senescence-associated secretory phenotype (SASP). There are two main types of cellular senescence, including telomere-dependent replicative senescence and stress-induced premature senescence. The process of cellular senescence is mainly controlled by two effector pathways, namely, the p53-p21 and p16-retinoblastoma protein (pRB) pathways. Vascular smooth muscle cells (VSMCs) are integral parts of arteries and play an important role in vascular structure and function. VSMC senescence may be triggered by many factors, such as angiotensin II, oxidative stress, inflammation, DNA damage, and small molecule compounds. These inducers are able to genetically and epigenetically regulate VSMC senescence. The senescence of VSMCs together with the SASP contributes to chronic vascular inflammation, the loss of arterial function, and the development of age-related diseases. Current evidence suggests that the senescence of VSMCs might be harmful to individual health, whereas its influence on the lifespan is not clear. The purpose of this paper was to review the current knowledge regarding VSMC senescence and its relevance to hypertension, atherosclerosis, and diabetes, as well as the potential mechanisms responsible for VSMC senescence in these age-related diseases.     

氧化应激诱导的细胞衰老过程中细胞生长相关基因的表达

应激诱导的细胞早衰与复制性细胞衰老有相似的细胞表型,但其机制不尽相同.分析二者的衰老相关基因表达特点对了解应激因素诱导细胞衰老的机制有重要意义. 本文对过氧化氢诱导的HeLa细胞早衰过程中的关键衰老相关基因及其转录后调控因子的表达做了分析.结果发现,在复制性衰老过程中明显降低的cyclin A、cyclin B1、c-fos及HuR,在温和过氧化氢诱导的细胞早衰过程中并无明显改变;在氧化应激诱导的细胞早衰过程中,p21与p16表达升高,AUF1则降低,与复制性衰老过程一致;p21 mRNA半衰期在复制性衰老过程中无明显变化,但在氧化应激诱导的细胞早衰过程中则显著延长.上述结果提示,尽管氧化应激诱导的细胞早衰与复制性衰老存在相似基因表达变化,调控机制则不尽相同.     

Cell-based screen for altered nuclear phenotypes reveals senescence progression in polyploid cells after Aurora kinase B inhibition

Cellular senescence is a widespread stress response and is widely considered to be an alternative cancer therapeutic goal. Unlike apoptosis, senescence is composed of a diverse set of subphenotypes, depending on which of its associated effector programs are engaged. Here we establish a simple and sensitive cell-based prosenescence screen with detailed validation assays. We characterize the screen using a focused tool compound kinase inhibitor library. We identify a series of compounds that induce different types of senescence, including a unique phenotype associated with irregularly shaped nuclei and the progressive accumulation of G1 tetraploidy in human diploid fibroblasts. Downstream analyses show that all of the compounds that induce tetraploid senescence inhibit Aurora kinase B (AURKB). AURKB is the catalytic component of the chromosome passenger complex, which is involved in correct chromosome alignment and segregation, the spindle assembly checkpoint, and cytokinesis. Although aberrant mitosis and senescence have been linked, a specific characterization of AURKB in the context of senescence is still required. This proof-of-principle study suggests that our protocol is capable of amplifying tetraploid senescence, which can be observed in only a small population of oncogenic RAS-induced senescence, and provides additional justification for AURKB as a cancer therapeutic target.     

Heat shock induces apoptosis in human embryonic stem cells but a premature senescence phenotype in their differentiated progeny

Embryonic stem cells (ESC) are able to self-renew and to differentiate into any cell type. To escape error transmission to future cell progeny, ESC require robust mechanisms to ensure genomic stability. It was stated that stress defense of mouse and human ESC against oxidative stress and irradiation is superior compared with differentiated cells. Here, we investigated heat shock response of human ESC (hESC) and their differentiated progeny. Fibroblast-like cells were generated by spontaneous hESC differentiation via embryoid bodies. Like normal human diploid fibroblasts, these cells have a finite lifespan in culture, undergo replicative senescence and die. We found that sublethal heat shock affected survival of both cell types, but in hESC it induced apoptosis, whereas in differentiated cells it produced cell cycle arrest and premature senescence phenotype. Heat shock survived hESC and differentiated cells restored the properties of initial cells. Heated hESC progeny exhibited pluripotent markers and the capacity to differentiate into the cells of three germ layers. Fibroblast-like cells resisted heat shock, proliferated for a limited number of passages and entered replicative senescence as unheated parental cells. Taken together, these results show for the first time that both hESC and their differentiated derivatives are sensitive to heat shock, but the mechanisms of their stress response are different: hESC undergo apoptosis, whereas differentiated cells under the same conditions exhibit stress-induced premature senescence (SIPS) phenotype. Both cell types that survived sublethal heat shock sustain parental cell properties.     

Heat shock induces apoptosis in human embryonic stem cells but a premature senescence phenotype in their differentiated progeny

Embryonic stem cells (ESC) are able to self-renew and to differentiate into any cell type. To escape error transmission to future cell progeny, ESC require robust mechanisms to ensure genomic stability. It was stated that stress defense of mouse and human ESC against oxidative stress and irradiation is superior compared with differentiated cells. Here, we investigated heat shock response of human ESC (hESC) and their differentiated progeny. Fibroblast-like cells were generated by spontaneous hESC differentiation via embryoid bodies. Like normal human diploid fibroblasts, these cells have a finite lifespan in culture, undergo replicative senescence and die. We found that sublethal heat shock affected survival of both cell types, but in hESC it induced apoptosis, whereas in differentiated cells it produced cell cycle arrest and premature senescence phenotype. Heat shock survived hESC and differentiated cells restored the properties of initial cells. Heated hESC progeny exhibited pluripotent markers and the capacity to differentiate into the cells of three germ layers. Fibroblast-like cells resisted heat shock, proliferated for a limited number of passages and entered replicative senescence as unheated parental cells. Taken together, these results show for the first time that both hESC and their differentiated derivatives are sensitive to heat shock, but the mechanisms of their stress response are different: hESC undergo apoptosis, whereas differentiated cells under the same conditions exhibit stress-induced premature senescence (SIPS) phenotype. Both cell types that survived sublethal heat shock sustain parental cell properties.     

Ras Regulates Rb via NORE1A

Mutations in the Ras oncogene are one of the most frequent events in human cancer. Although Ras regulates numerous growth-promoting pathways to drive transformation, it can paradoxically promote an irreversible cell cycle arrest known as oncogene-induced senescence. Although senescence has clearly been implicated as a major defense mechanism against tumorigenesis, the mechanisms by which Ras can promote such a senescent phenotype remain poorly defined. We have shown recently that the Ras death effector NORE1A plays a critical role in promoting Ras-induced senescence and connects Ras to the regulation of the p53 tumor suppressor. We now show that NORE1A also connects Ras to the regulation of a second major prosenescent tumor suppressor, the retinoblastoma (Rb) protein. We show that Ras induces the formation of a complex between NORE1A and the phosphatase PP1A, promoting the activation of the Rb tumor suppressor by dephosphorylation. Furthermore, suppression of Rb reduces NORE1A senescence activity. These results, together with our previous findings, suggest that NORE1A acts as a critical tumor suppressor node, linking Ras to both the p53 and the Rb pathways to drive senescence.     

A Model for p38MAPK-Induced Astrocyte Senescence

Experimental evidence indicates that aging leads to accumulation of senescent cells in tissues and they develop a secretory phenotype (also known as SASP, for senescence-associated secretory phenotype) that can contribute to chronic inflammation and diseases. Recent results have showed that markers of senescence in astrocytes from aged brains are increased in brains with Alzheimer’s disease. These studies strongly involved the stress kinase p38MAPK in the regulation of the secretory phenotype of astrocytes, yet the molecular mechanisms underlying the onset of senescence and SASP activation remain unclear. In this work, we propose a discrete logical model for astrocyte senescence determined by the level of DNA damage (reparable or irreparable DNA strand breaks) where the kinase p38MAPK plays a central role in the regulation of senescence and SASP. The model produces four alternative stable states: proliferation, transient cycle arrest, apoptosis and senescence (and SASP) computed from its inputs representing DNA damages. Perturbations of the model were performed through gene gain or loss of functions and compared with results concerning cultures of normal and mutant astrocytes showing agreement in most cases. Moreover, the model allows some predictions that remain to be tested experimentally.     

Exploiting tumor cell senescence in anticancer therapy

Cellular senescence is a physiological process of irreversible cell-cycle arrest that contributes to various physiological and pathological processes of aging. Whereas replicative senescence is associated with telomere attrition after repeated cell division, stress-induced premature senescence occurs in response to aberrant oncogenic signaling, oxidative stress, and DNA damage which is independent of telomere dysfunction. Recent evidence indicates that cellular senescence provides a barrier to tumorigenesis and is a determinant of the outcome of cancer treatment. However, the senescence-associated secretory phenotype, which contributes to multiple facets of senescent cancer cells, may influence both cancer-inhibitory and cancer-promoting mechanisms of neighboring cells. Conventional treatments, such as chemo- and radiotherapies, preferentially induce premature senescence instead of apoptosis in the appropriate cellular context. In addition, treatment-induced premature senescence could compensate for resistance to apoptosis via alternative signaling pathways. Therefore, we believe that an intensive effort to understand cancer cell senescence could facilitate the development of novel therapeutic strategies for improving the efficacy of anticancer therapies. This review summarizes the current understanding of molecular mechanisms, functions, and clinical applications of cellular senescence for anticancer therapy. [BMB Reports 2014; 47(2): 51-59]     

Epigenetic control of cellular senescence in disease: opportunities for therapeutic intervention

Understanding how senescence is established and maintained is an important area of study both for normal cell physiology and in tumourigenesis. Modifications to N-terminal tails of histone proteins, which can lead to chromatin remodelling, appear to be key to the regulation of the senescence phenotype. Epigenetic mechanisms such as modification of histone proteins have been shown to be sufficient to regulate gene expression levels and specific gene promoters can become epigenetically altered at senescence. This suggests that epigenetic mechanisms are important in senescence and further suggests epigenetic deregulation could play an important role in the bypass of senescence and the acquisition of a tumourigenic phenotype. Tumour suppressor proteins and cellular senescence are intimately linked and such proteins are now known to regulate gene expression through chromatin remodelling, again suggesting a link between chromatin modification and cellular senescence. Telomere dynamics and the expression of the telomerase genes are also both implicitly linked to senescence and tumourigenesis, and epigenetic deregulation of the telomerase gene promoters has been identified as a possible mechanism for the activation of telomere maintenance mechanisms in cancer. Recent studies have also suggested that epigenetic deregulation in stem cells could play an important role in carcinogenesis, and new models have been suggested for the attainment of tumourigenesis and bypass of senescence. Overall, proper regulation of the chromatin environment is suggested to have an important role in the senescence pathway, such that its deregulation could lead to tumourigenesis.     

Decreased skp2 expression is necessary but not sufficient for therapy-induced senescence in prostate cancer

Therapy-induced senescence (TIS), a cytostatic stress response in cancer cells, is induced inefficiently by current anticancer agents and radiation. The mechanisms that mediate TIS in cancer cells are not well defined. Herein, we characterize a robust senescence response both in vitro and in vivo to the quinone diaziquone (AZQ), previously identified in a high-throughput senescence-induction small-molecule screen. Using AZQ and several other agents that induce senescence, we screened a series of cyclin-dependent kinase inhibitors and found that p27(Kip1) was induced in all investigated prostate cancer cell lines. The ubiquitin-ligase Skp2 negatively regulates p27(Kip1) and, during TIS, is translocated to the cytoplasm before its expression is decreased in senescent cells. Overexpression of Skp2 blocks the effects of AZQ on senescence and p27(Kip1) induction. We also find that stable long-term short hairpin RNA knockdown of Skp2 decreases proliferation but does not generate the complete senescence phenotype. We conclude that Skp2 participates in regulating TIS but, alone, is insufficient to induce senescence in cancer cells.     

From cell senescence to age-related diseases: differential mechanisms of action of senescence-associated secretory phenotypes

Cellular senescence is a process by which cells enter a state of permanent cell cycle arrest. It is commonly believed to underlie organismal aging and age-associated diseases. However, the mechanism by which cellular senescence contributes to aging and age-associated pathologies remains unclear. Recent studies showed that senescent cells exert detrimental effects on the tissue microenvironment, generating pathological facilitators or aggravators. The most significant environmental effector resulting from senescent cells is the senescence-associated secretory phenotype (SASP), which is constituted by a strikingly increased expression and secretion of diverse pro-inflammatory cytokines. Careful investigation into the components of SASPs and their mechanism of action, may improve our understanding of the pathological backgrounds of age-associated diseases. In this review, we focus on the differential expression of SASP-related genes, in addition to SASP components, during the progress of senescence. We also provide a perspective on the possible action mechanisms of SASP components, and potential contributions of SASP-expressing senescent cells, to age-associated pathologies. [BMB Reports 2015; 48(10): 549-558]     

Hybrids from fusion of normal human T lymphocytes with immortal human cells exhibit limited life span

A number of normal human cell types have been shown to exhibit cellular senescence in vitro. We and others had found that fusion of normal human fibroblasts with immortal human cells yielded hybrids having limited lifespan. This indicated that the phenotype of cellular senescence is dominant and that immortality results from recessive changes in genes involved in growth control. They also supported the hypothesis that senescence results from genetic mechanisms rather than random damage. Since T lymphocytes are a highly differentiated cell type, in contrast to fibroblasts, it was of interest to determine whether similar mechanisms caused senescence in the T cells. We therefore fused normal human T lymphocytes with an immortal human cell line to determine whether they could restore the senescent, nondividing phenotype in hybrids, as do normal human fibroblasts. Eleven of fifteen hybrid clones studied exhibited limited proliferative potential after achieving a range of population doubling similar to that observed in the cell fusion studies involving normal fibroblasts. These results provide evidence that cellular senescence in T lymphocytes occurs via genetic mechanisms.     

The impact of autophagy on the development of senescence in primary tubular epithelial cells

Autophagy and senescence are 2 distinct pathways that are importantly involved in acute kidney injury and renal repair. Recent data indicate that the 2 processes might be interrelated. To investigate the potential link between autophagy and senescence in the kidney we isolated primary tubular epithelial cells (PTEC) from wild-type mice and monitored the occurrence of cellular senescence during autophagy activation and inhibition. We found that the process of cell isolation and transfer into culture was associated with a strong basal autophagic activation in PTEC. Specific inhibition of autophagy by silencing autophagy-related 5 (Atg5) counteracted the occurrence of senescence hallmarks under baseline conditions. Reduced senescent features were also observed in Atg5 silenced PTEC after γ-irradiation and during H-Ras induced oncogenic senescence, but the response was less uniform in these stress models. Senescence inhibition was paralleled by better preservation of a mature epithelial phenotype in PTEC. Interestingly, treatment with rapamycin, which acts as an activator of autophagy, also counteracted the occurrence of senescence features in PTEC. While we interpret the anti-senescent effect of rapamycin as an autophagy-independent effect of mTOR-inhibition, the more specific approach of Atg5 silencing indicates that overactivated autophagy can have pro-senescent effects in PTEC. These results highlight the complex interaction between cell culture dependent stress mechanisms, autophagy and senescence.     

Pathway analysis of senescence-associated miRNA targets reveals common processes to different senescence induction mechanisms

Multiple mechanisms of senescence induction exist including telomere attrition, oxidative stress, oncogene expression and DNA damage signalling. The regulation of the cellular changes required to respond to these stimuli and create the complex senescent cell phenotype has many different mechanisms. MiRNAs present one mechanism by which genes with diverse functions on multiple pathways can be simultaneously regulated. In this study we investigated 12 miRNAs previously identified as senescence regulators. Using pathway analysis of their target genes we tested the relevance of miRNA regulation in the induction of senescence. Our analysis highlighted the potential of these senescence-associated miRNAs (SA-miRNAs) to regulate the cell cycle, cytoskeletal remodelling and proliferation signalling logically required to create a senescent cell. The reanalysis of publicly available gene expression data from studies exploring different senescence stimuli also revealed their potential to regulate core senescence processes, regardless of stimuli. We also identified stimulus specific apoptosis survival pathways theoretically regulated by the SA-miRNAs. Furthermore the observation that miR-499 and miR-34c had the potential to regulate all 4 of the senescence induction types we studied highlights their future potential as novel drug targets for senescence induction.     

The antioxidative defense system is involved in the delayed senescence in a wheat mutant tasg1

Wheat, which is the most important food crop worldwide, is a cereal that presents considerable potential for increased yield. A new wheat (Triticum aestivum L.) mutant tasg1 with delayed leaf senescence was constructed using ethyl methane sulfonate as a mutagen. Natural senescence in tasg1 was distinctly delayed in the field, as indicated by the slower progression of chlorophyll degradation, net photosynthetic rate than its wild type. Further, the malondialdehyde and the hydrogen peroxide content was lower and antioxidative enzyme activity higher in tasg1 than those in its wild type during both natural senescence and methyl viologen-induced oxidative stress. The results suggest that tasg1 is a functional stay-green wheat mutant with the Type B (in which senescence initiates on schedule, but progresses at a rate lower than that in the respective WTs) or Type A (in which senescence initiates late but proceeds at a normal rate) and B combination and that the competence of the antioxidant defense system is one of the most important mechanisms underlying the expression of the stay-green phenotype.     

Aging of the cells: Insight into cellular senescence and detection Methods

Cellular theory of aging states that human aging is the result of cellular aging, in which an increasing proportion of cells reach senescence. Senescence, from the Latin word senex, means “growing old,” is an irreversible growth arrest which occurs in response to damaging stimuli, such as DNA damage, telomere shortening, telomere dysfunction and oncogenic stress leading to suppression of potentially dysfunctional, transformed, or aged cells. Cellular senescence is characterized by irreversible cell cycle arrest, flattened and enlarged morphology, resistance to apoptosis, alteration in gene expression and chromatin structure, expression of senescence associated- β-galactosidase (SA-β-gal) and acquisition of senescence associated secretory phenotype (SASP). In this review paper, different types of cellular senescence including replicative senescence (RS) which occurs due to telomere shortening and stress induced premature senescence (SIPS) which occurs in response to different types of stress in cells, are discussed. Biomarkers of cellular senescence and senescent assays including BrdU incorporation assay, senescence associated- β-galactosidase (SA-β-gal) and senescence-associated heterochromatin foci assays to detect senescent cells are also addressed.     

Proteasome modulates mitochondrial function during cellular senescence

Proteasome plays fundamental roles in the removal of oxidized proteins and in the normal degradation of short-lived proteins. Previously we have provided evidence that the impairment in proteasome observed during the replicative senescence of human fibroblasts has significant effects on MAPK signaling, proliferation, life span, senescent phenotype, and protein oxidative status. These studies have demonstrated that proteasome inhibition and replicative senescence caused accumulation of intracellular protein carbonyl content. In this study, we have investigated the mechanisms by which proteasome dysfunction modulates protein oxidation during cellular senescence. The results indicate that proteasome inhibition during replicative senescence has significant effects on intra- and extracellular ROS production in vitro. The data also show that ROS impaired the proteasome function, which is partially reversible by antioxidants. Increases in ROS after proteasome inhibition correlated with a significant negative effect on the activity of most mitochondrial electron transporters. We propose that failures in proteasome during cellular senescence lead to mitochondrial dysfunction, ROS production, and oxidative stress. Furthermore, it is likely that changes in proteasome dynamics could generate a prooxidative condition at the immediate extracellular microenvironment that could cause tissue injury during aging, in vivo.     

Regulation of apoptosis and replicative senescence in CD8+ T cell following acute viral infection

Viral infections are characterised by a large expansion of CD8⁺ effector T cells. Once generated, these T cells must be cleared and homeostasis re-established. In this review we describe two mechanisms, apoptosis and replicative senescence which are thought to play a vital role in this process. Apoptosis clears the unwanted T cells at the end of an immune response whereas replicative senescence prevents unlimited expansion of T cells. For successful memory to be produced T cells must avoid apoptosis and avoid replicative senescence.     

Cellular senescence and cancer treatment

Cellular senescence, an irreversible cell-cycle arrest, reflects a safeguard program that limits the proliferative capacity of the cell exposed to endogenous or exogenous stress signals. A number of recent studies have clarified that an acutely inducible form of cellular senescence may act in response to oncogenic activation as a natural barrier to interrupt tumorigenesis at a premalignant level. Paralleling the increasing insights into premature senescence as a tumor suppressor mechanism, a growing line of evidence identifies cellular senescence as a critical effector program in response to DNA damaging chemotherapeutic agents. This review discusses molecular pathways to stress-induced senescence, the interference of a terminal arrest condition with clinical outcome, and the critical overlap between premature senescence and apoptosis as both tumor suppressive and drug-responsive cellular programs.