Therapeutic Opportunities Presented by Modulation of Cellular Senescence.

Cellular senescence is a permanent state of growth arrest coupled with profound changes in phenotype that can be triggered by multiple extrinsic or intrinsic stimuli. Senescence is a process-level example of the evolution of ageing mechanisms through antagonistic pleiotropy and plays a primary role in tumour suppression, although evidence is mounting for its involvement in other fundamental physiological processes. Evidence from human premature ageing diseases and from transgenic mice in which it is possible to specifically delete senescent cells is consistent with a model in which the accumulation of senescent cells through the life course is responsible for later life chronic disease and impairment. The removal of senescent cells or their reversion to a phenotypically benign state is thus an important emerging goal of translational medicine.Modern bioinformatic approaches based on text mining have compiled co-mentions of cell senescence and age-related diseases allowing an impartial ranking of the impairments most closely associated with this process. Following this schema, the evidence for the involvement of senescence in several highly ranked pathologies is reviewed, alongside potential methods for the ablation of senescent cells or their reversion to their primary phenotype with polyphenolics or inhibitors of p38 MAP kinase. Lastly, the potential for senescence to act as a barrier to the development of bioartificial organs designed to treat some of these conditions is discussed.

Effects of resveratrol and its analogues on the cell cycle of equine mesenchymal stem/stromal cells.

Resveratrol (RSV; trans-3,5,4'-trihydroxystilbene) strongly activates sirtuin 1, and it and its analogue V29 enhance the proliferation of mesenchymal stem/stromal cells (MSCs).Although culture medium containing 5-azacytydine and RSV inhibits senescence of adipose tissue-derived MSCs isolated from horses with metabolic syndrome, few studies have reported the effects of RSV on equine bone marrow-derived MSCs (eBMMSCs) isolated from horses without metabolic syndrome. The aim of this study was to investigate the effects of RSV and V29 on the cell cycle of eBMMSCs. Following treatment with 5 µM RSV or 10 µM V29, the cell proliferation capacity of eBMMSCs derived from seven horses was evaluated by EdU (5-ethynyl-2'-deoxyuridine) and Ki-67 antibody assays. Brightfield images of cells and immunofluorescent images of EdU, Ki-67, and DAPI staining were recorded by fluorescence microscopy, and the number of cells positive for each was quantified and compared by Friedman's test at P<0.05. The growth fraction of eBMMSCs was significantly increased by RSV and V29 as measured by the EdU assay (control 28.1% ± 13.8%, V29 31.8% ± 14.6%, RSV 32.0% ± 10.8%; mean ± SD; P<0.05) but not as measured by the Ki-67 antibody assay (control 27.0% ± 11.2%, V29 27.4% ± 10.8%, RSV 27.7% ± 6.8%). RSV and V29 promoted progression of the cell cycle of eBMMSCs into the S phase and may be useful for eBMMSC expansion.

FOXO1 and ETV6 genes may represent novel regulators of splicing factor expression in cellular senescence.

Cellular plasticity is a key facet of cellular homeostasis requiring correct temporal and spatial patterns of alternative splicing. Splicing factors, which orchestrate this process, demonstrate age-related dysregulation of expression; they are emerging as potential influences on aging and longevity. The upstream drivers of these alterations are still unclear but may involve aberrant cellular signaling. We compared the phosphorylation status of proteins in multiple signaling pathways in early and late passage human primary fibroblasts. We then assessed the impact of chemical inhibition or targeted knockdown of direct downstream targets of the ERK and AKT pathways on splicing factor expression, cellular senescence, and proliferation kinetics in senescent primary human fibroblasts. Components of the ERK and AKT signaling pathways demonstrated altered activation during cellular aging. Inhibition of AKT and ERK pathways led to up-regulation of splicing factor expression, reduction in senescent cell load, and partial reversal of multiple cellular senescence phenotypes in a dose-dependent manner. Furthermore, targeted knockdown of the genes encoding the downstream targets FOXO1 or ETV6 was sufficient to mimic these observations. Our results suggest that age-associated dysregulation of splicing factor expression and cellular senescence may derive in part from altered activity of ERK and AKT signaling and may act in part through the ETV6 and FOXO1 transcription factors. Targeting the activity of downstream effectors of ERK and AKT may therefore represent promising targets for future therapeutic intervention.-Latorre, E., Ostler, E. L., Faragher, R. G. A., Harries, L. W. FOXO1 and ETV6 genes may represent novel regulators of splicing factor expression in cellular senescence.

Novel resveratrol derivatives have diverse effects on the survival, proliferation and senescence of primary human fibroblasts.

Resveratrol alters the cytokinetics of mammalian cell populations in a dose dependent manner. Concentrations above 25-50 µM typically trigger growth arrest, senescence and/or apoptosis in multiple different cell types. In contrast, concentrations below 10 µM enhance the growth of log phase cell cultures and can rescue senescence in multiple strains of human fibroblasts. To better understand the structural features that regulate these effects, a panel of 24 structurally-related resveralogues were synthesised and evaluated for their capacity to activate SIRT1, as determined by an ex-vivo SIRT1 assay, their toxicity, as measured by lactate dehydrogenase release, and their effects on replicative senescence in MRC5 human fibroblasts as measured by their effects on Ki67 immunoreactivity and senescence-associated ß galactosidase activity. Minor modifications to the parent stilbene, resveratrol, significantly alter the biological activities of the molecules. Replacement of the 3,5-dihydroxy substituents with 3,5-dimethoxy groups significantly enhances SIRT1 activity, and reduces toxicity. Minimising other strong conjugative effects also reduces toxicity, but negatively impacts SIRT1 activation. At 100 µM many of the compounds, including resveratrol, induce senescence in primary MRC5 cells in culture. Modifications that reduce or remove this effect match those that reduce toxicity leading to a correlation between reduction in labelling index and increase in LDH release. At 10 µM, the majority of our compounds significantly enhance the growth fraction of log phase cultures of MRC5 cells, consistent with the rescue of a subpopulation of cells within the culture from senescence. SIRT1 activation is not required for rescue to occur but enhances the size of the effect.

Resveralogues: From Novel Ageing Mechanisms to New Therapies?

For much of the 20th century the ageing process was thought to be the result of the interplay of many different biological processes, each with relatively small effects on organismal lifespan. However, this model is no longer tenable. Rather it seems a few biological mechanisms, including nutrient sensing, telomere attrition and cellular senescence, mediate large effects on health and longevity. Biogerontology may have suffered from initial delusions of complexity. However, we argue that it is premature to assume either that the list of biological processes influencing lifespan is now comprehensive or that these mechanisms act independently of each other. A case in point is provided by recent work linking together changes in RNA splicing with advancing age and the ability of polyphenolics based on resveratrol to reverse replicative senescence. In this opinion piece, we propose a novel model in which the factors regulating splice restriction and those controlling cell senescence intersect across chronological and divisional time, giving rise to senescent and growing cells with more diverse properties than previously thought. We also consider therapeutic opportunities and potential problems in the light of this revised conceptual understanding of human cell senescence and ageing.

Telomere elongation through hTERT immortalization leads to chromosome repositioning in control cells and genomic instability in Hutchinson-Gilford progeria syndrome fibroblasts, expressing a novel SUN1 isoform.

Immortalizing primary cells with human telomerase reverse transcriptase (hTERT) has been common practice to enable primary cells to be of extended use in the laboratory because they avoid replicative senescence. Studying exogenously expressed hTERT in cells also affords scientists models of early carcinogenesis and telomere behavior. Control and the premature ageing disease-Hutchinson-Gilford progeria syndrome (HGPS) primary dermal fibroblasts, with and without the classical G608G mutation have been immortalized with exogenous hTERT. However, hTERT immortalization surprisingly elicits genome reorganization not only in disease cells but also in the normal control cells, such that whole chromosome territories normally located at the nuclear periphery in proliferating fibroblasts become mislocalized in the nuclear interior. This includes chromosome 18 in the control fibroblasts and both chromosomes 18 and X in HGPS cells, which physically express an isoform of the LINC complex protein SUN1 that has previously only been theoretical. Additionally, this HGPS cell line has also become genomically unstable and has a tetraploid karyotype, which could be due to the novel SUN1 isoform. Long-term treatment with the hTERT inhibitor BIBR1532 enabled the reduction of telomere length in the immortalized cells and resulted that these mislocalized internal chromosomes to be located at the nuclear periphery, as assessed in actively proliferating cells. Taken together, these findings reveal that elongated telomeres lead to dramatic chromosome mislocalization, which can be restored with a drug treatment that results in telomere reshortening and that a novel SUN1 isoform combined with elongated telomeres leads to genomic instability. Thus, care should be taken when interpreting data from genomic studies in hTERT-immortalized cell lines.

Obesity and type-2 diabetes as inducers of premature cellular senescence and ageing.

Cellular senescence is now considered as a major mechanism in the development and progression of various diseases and this may include metabolic diseases such as obesity and type-2 diabetes. The presence of obesity and diabetes is a major risk factor in the development of additional health conditions, such as cardiovascular disease, kidney disease and cancer. Since senescent cells can drive disease development, obesity and diabetes can potentially create an environment that accelerates cell senescence within other tissues of the body. This can consequently manifest as age-related biological impairments and secondary diseases. Cell senescence in cell types linked with obesity and diabetes, namely adipocytes and pancreatic beta cells will be explored, followed by a discussion on the role of obesity and diabetes in accelerating ageing through induction of premature cell senescence mediated by high glucose levels and oxidised low-density lipoproteins. Particular emphasis will be placed on accelerated cell senescence in endothelial progenitor cells, endothelial cells and vascular smooth muscle cells with relation to cardiovascular disease and proximal tubular cells with relation to kidney disease. A summary of the potential strategies for therapeutically targeting senescent cells for improving health is also presented.

Small molecule modulation of splicing factor expression is associated with rescue from cellular senescence.

BACKGROUND: Altered expression of mRNA splicing factors occurs with ageing in vivo and is thought to be an ageing mechanism. The accumulation of senescent cells also occurs in vivo with advancing age and causes much degenerative age-related pathology. However, the relationship between these two processes is opaque. Accordingly we developed a novel panel of small molecules based on resveratrol, previously suggested to alter mRNA splicing, to determine whether altered splicing factor expression had potential to influence features of replicative senescence. RESULTS: Treatment with resveralogues was associated with altered splicing factor expression and rescue of multiple features of senescence. This rescue was independent of cell cycle traverse and also independent of SIRT1, SASP modulation or senolysis. Under growth permissive conditions, cells demonstrating restored splicing factor expression also demonstrated increased telomere length, re-entered cell cycle and resumed proliferation. These phenomena were also influenced by ERK antagonists and agonists. CONCLUSIONS: This is the first demonstration that moderation of splicing factor levels is associated with reversal of cellular senescence in human primary fibroblasts. Small molecule modulators of such targets may therefore represent promising novel anti-degenerative therapies.

Suppression of the senescence-associated secretory phenotype (SASP) in human fibroblasts using small molecule inhibitors of p38 MAP kinase and MK2.

Senescent cells show an altered secretome profile termed the senescence-associated secretory phenotype (SASP). There is an increasing body of evidence that suggests that the accumulation of SASP-positive senescent cells in humans is partially causal in the observed shift to a low-level pro-inflammatory state in aged individuals. This in turn suggests the SASP as a possible therapeutic target to ameliorate inflammatory conditions in the elderly, and thus a better understanding of the signalling pathways underlying the SASP are required. Prior studies using the early generation p38 MAPK inhibitor SB203580 indicated that p38 signalling was required for the SASP. In this study, we extend these observations using two next-generation p38 inhibitors (UR-13756 and BIRB 796) that have markedly improved selectivity and specificity compared to SB203580, to strengthen the evidence that the SASP is p38-dependent in human fibroblasts. BIRB 796 has an efficacy and toxicity profile that has allowed it to reach Phase III clinical trials, suggesting its possible use to suppress the SASP in vivo. We also demonstrate for the first time a requirement for signalling through the p38 downstream MK2 kinase in the regulation of the SASP using two MK2 inhibitors. Finally, we demonstrate that a commercially-available multiplex cytokine assay technology can be used to detect SASP components in the conditioned medium of cultured fibroblasts from both young and elderly donors. This assay is a high-throughput, multiplex microtitre-based assay system that is highly sensitive, with very low sample requirements, allowing it to be used for low-volume human biological fluids. Our initial studies using existing multiplex plates form the basis for a "SASP signature" assay that could be used as a high-throughput system in a clinical study setting. Our findings therefore provide important steps towards the study of, and intervention in, the SASP in human ageing and age-related disease.

Recapitulation of Werner syndrome sensitivity to camptothecin by limited knockdown of the WRN helicase/exonuclease.

WRN is a RecQ helicase with an associated exonuclease activity important in DNA metabolism, including DNA replication, repair and recombination. In humans, deficiencies in WRN function cause the segmental progeroid Werner syndrome (WS), in which patients show premature onset of many hallmarks of normal human ageing. At the cellular level, WRN loss results in rapid replicative senescence, chromosomal instability and sensitivity to various DNA damaging agents including the topoisomerase inhibitor, camptothecin (CPT). Here, we investigate the potential of using either transient or stable WRN knockdown as a means of sensitising cells to CPT. We show that targeting WRN mRNA for degradation by either RNAi or hammerhead ribozyme catalysis renders human fibroblasts as sensitive to CPT as fibroblasts derived from WS patients, and furthermore, we find altered cell cycle transit and nucleolar destabilisation in these cells following CPT treatment. Such WS-like phenotypes are observed despite very limited decreases in total WRN protein, suggesting that levels of WRN protein are rate-limiting for the cellular response to camptothecin. These findings have major implications for development of anti-WRN agents that may be useful in sensitising tumour cells to clinically relevant topoisomerase inhibitors.

Simple Detection Methods for Senescent Cells: Opportunities and Challenges.

Cellular senescence, the irreversible growth arrest of cells from conditional renewal populations combined with a radical shift in their phenotype, is a hallmark of ageing in some mammalian species. In the light of this, interest in the detection of senescent cells in different tissues and different species is increasing. However much of the prior work in this area is heavily slanted towards studies conducted in humans and rodents; and in these species most studies concern primary fibroblasts or cancer cell lines rendered senescent through exposure to a variety of stressors. Complex techniques are now available for the detailed analysis of senescence in these systems. But, rather than focussing on these methods this review instead examines techniques for the simple and reproducible detection of senescent cells. Intended primary for the non-specialist who wishes to quickly detect senescent cells in tissues or species which may lack a significant evidence base on the phenomenon it emphasises the power of the original techniques used to demonstrate the senescence of cells, their interrelationship with other markers and their potential to inform on the senescent state in new species and archival specimens.

Cell divisions and mammalian aging: integrative biology insights from genes that regulate longevity.

Despite recent progress in the identification of genes that regulate longevity, aging remains a mysterious process. One influential hypothesis is the idea that the potential for cell division and replacement are important factors in aging. In this work, we review and discuss this perspective in the context of interventions in mammals that appear to accelerate or retard aging. Rather than focus on molecular mechanisms, we interpret results from an integrative biology perspective of how gene products affect cellular functions, which in turn impact on tissues and organisms. We review evidence suggesting that mutations that give rise to features resembling premature aging tend to be associated with cellular phenotypes such as increased apoptosis or premature replicative senescence. In contrast, many interventions in mice that extend lifespan and might delay aging, including caloric restriction, tend to either hinder apoptosis or result in smaller animals and thus may be the product of fewer cell divisions. Therefore, it appears plausible that changes in the number of times that cells, and particularly stem cells, divide during an organism's lifespan influence longevity and aging. We discuss possible mechanisms related to this hypothesis and propose experimental paradigms.

Can we intervene in human ageing?

Ageing is a progressive failure of defence and repair processes that produces physiological frailty (the loss of organ reserve with age), loss of homeostasis and eventual death. Over the past ten years exceptional progress has been made in understanding both why the ageing process happens and the mechanisms that are responsible for it. The study of natural mutants that accelerate some, but not all, of the features of the human ageing process has now progressed to a degree that drug trials are either taking place or can be envisaged. Simultaneously, a series of mutations have been identified in different species that confer extended healthy life, indicating that the ageing process is much more malleable than might have been expected and that single interventions have the potential to delay the onset of multiple age-associated conditions. Data generated using these organisms have led to the formulation of a powerful new hypothesis, the 'green theory' of ageing. This proposes that a finite capacity to carry out broad-spectrum detoxification and recycling is the primary mechanistic limit on organismal lifespan. This is turn suggests important new experimental approaches and potential interventions designed to increase healthy lifespan.

A transcriptomic analysis of the EK1.Br strain of human fibroblastoid keratocytes: the effects of growth, quiescence and senescence.

There is a growing need within ocular research for well-defined cellular models of normal corneal biology. To meet this need we created and partially characterised a standard strain of human fibroblastoid keratocytes (EK1.Br) and demonstrated that phenotypic changes occur within these cells with replicative senescence in vitro. Using Affymetrix HG-U133A oligonucleotide arrays, this paper reports both a comprehensive analysis of the transcriptome of EK1.Br in the growing, quiescent and senescent states and a comparison of that transcriptome with those of primary corneal endothelium, lung fibroblasts and dermal fibroblasts grown under identical conditions. Data mining shows (i) that EK1.Br retain the characteristic transcriptional fingerprint of keratocytes in vitro (ii) that this phenotype can be distinguished from those of other 'fibroblasts' by groups of highly differentially expressed genes and (iii) that senescence induces a distinct dedifferentiation phenomenon in EK1.Br. These findings are contextualised into the broader literature on replicative senescence and are supported with a web-accessible and fully searchable public-access database (www.madras.cf.ac.uk/cornea).

A novel population of repair cells identified in the stroma of the human cornea.

The transmembrane protein CD133 is expressed on somatic stem cells of various adult human tissues. To investigate whether human corneal stroma also contains CD133-expressing cells and to analyze their functional features, stromal cells were isolated by collagenase digestion, immunophenotyped, and transferred to different culture systems to determine their stem cell properties as well as their differentiation potentials. For comparison, the embryonic keratocyte cell line EK1.Br, the dermal stromal cell line NHDF, and stromal cells of diseased corneas were studied. On average, 5.3% of the normal stromal cells expressed the stem cell marker CD133 and 3.6% co-expressed CD34. Expression of CD133 but not CD34 was also demonstrated for EK1.Br cells, whereas NHDF cells were negative for both markers. Further analysis of CD133(+) normal corneal cells revealed that a significant proportion displayed a monocytic phenotype with co-expression of CD45 and CD14. In diseased corneas, up to 26.8% of the stromal cells showed expression of CD133, and virtually all CD133(+) cells co-expressed CD14 but not CD45. Moreover, using a standard clonogenic assay, normal stromal cells had the capacity to form colonies of the macrophage lineage. These colonies could be further differentiated into lumican-expressing keratocytes. Our data suggest that the human corneal stroma harbors CD133(+) monocytic progenitor cells, which possess the potential to differentiate into the fibrocytic lineage. Thus, CD133(+) /CD45(+) /CD14(+) cells might represent stromal repair cells that differentiate into keratocytes via a CD133(+)/CD45()/CD14(+) intermediate stage. The findings from our study may shed new light on regenerative processes of the human corneal stroma.

Cyclin D1 overexpression permits the reproducible detection of senescent human vascular smooth muscle cells.

The senescence of mitotic cells is hypothesized to play a causal role in organismal aging. Cultures of normal human cells become senescent in vitro as a result of a continuous decline in the mitotic fraction from cell turnover. However, one potential barrier to the evaluation of the frequency and distribution of senescent cells in tissues is the absence of a panel of robust markers for the senescent state. In parallel with an analysis of the growth kinetics of human vascular smooth muscle cells, we have undertaken transcriptomic comparisons of early- and late-passage cultures of human vascular smooth muscle cells to identify potential markers that can distinguish between senescent and growth-competent cells. A wide range of genes are upregulated at senescence in human vascular smooth muscle cells. In particular, we have identified a 12-fold upregulation of expression in the cyclin D1 message, which is reflected in a concomitant upregulation at the protein level. Quantitative cytochemical analysis of senescent and growing vascular smooth muscle cells indicates that cyclin D1 reactivity is a considerably better marker of replicative senescence than senescence-associated beta-galactosidase activity. We have applied this new marker (in combination with Ki67, COMET, and TUNEL staining) to the study of human vascular smooth muscle cells treated with resveratrol, a putative anti-aging molecule known to have significant effects on cell growth.

alpha-Lipoic acid and glutathione protect against the prooxidant activity of SOD/catalase mimetic manganese salen derivatives.

Manganese(III) N,N'-ethylenebis(salicylideneiminato) chloride (Mn-salen chloride) and manganese(III) N,N'-ethylenebis(3-methoxysalicylideneiminato) chloride (Mn-(3,3'-MeO)salen chloride) are in vitro superoxide dismutase and catalase mimetics. They protect against free radical-related disease in animals, but Mn-salen can also be a potent prooxidant, damaging free DNA. Mn-salen protects human fibroblast DNA against hydrogen peroxide damage, however, damage to free DNA was confirmed by the comet assay. The DNA-damaging activity was dramatically reduced by co-administration with glutathione with the combination being less damaging to free DNA than either molecule alone. alpha-Lipoic acid, an antioxidant disulfide commonly used as a dietary supplement, also prevented Mn-salen prooxidant activity. Mn-(3,3'-MeO)salen protected fibroblasts against hydrogen peroxide as efficiently as Mn-salen and showed little damaging activity against free DNA. Protection was invested by both complexes in the presence and in the absence of EDTA, a potential competing chelator. Stabilities of the complexes with respect to decomposition and inactivation were studied by spectroscopic and electrochemical techniques. The complexes' binding to, and cleavage of, DNA was measured using a quartz crystal resonant sensor. Mn-salen was shown to bind strongly to DNA, prior to cleaving it; Mn-(3,3'-MeO)salen bound weakly and left DNA intact. Co-administration of either glutathione or alpha-lipoic acid appears to inhibit binding by Mn-salen thus preventing DNA-cleavage.

Absence of premature senescence in Werner's syndrome keratinocytes.

Werner's syndrome (WS) is an autosomal recessive genetic disorder caused by loss of function mutation in wrn and is a useful model of premature in vivo ageing. Cellular senescence is a plausible causal mechanism of mammalian ageing and, at the cellular level, WS fibroblasts show premature senescence resulting from a combination of telomeric attrition and replication fork stalling. Over 90% of WS fibroblast cultures achieve <20 population doublings (PD) in vitro compared to wild type human fibroblast cultures. It has been proposed that some cell types, capable of proliferation, will fail to show a premature senescence phenotype in response to wrn mutations. To test this hypothesis, human dermal keratinocytes (derived from both WS and wild type patients) were cultured long term. WS Keratinocytes showed a replicative lifespan in excess of 100 population doublings but maintained functional growth arrest mechanisms based on p16 and p53. The karyotype of the cells was superficially normal and the cultures retained markers characteristic of keratinocyte holoclones (stem cells) including p63 expression and telomerase activity. Accordingly we conclude that, in contrast to WS fibroblasts, WS keratinocytes do not demonstrate slow growth rates or features of premature senescence. These findings suggest that the epidermis is among the tissue types that do not display symptoms of premature ageing caused by loss of function of wrn. This is in support that Werner's syndrome is a segmental progeroid syndrome.