Cellular senescence: from growth arrest to immunogenic conversion.

Cellular senescence was first reported in human fibroblasts as a state of stable in vitro growth arrest following extended culture. Since that initial observation, a variety of other phenotypic characteristics have been shown to co-associate with irreversible cell cycle exit in senescent fibroblasts. These include (1) a pro-inflammatory secretory response, (2) the up-regulation of immune ligands, (3) altered responses to apoptotic stimuli and (4) promiscuous gene expression (stochastic activation of genes possibly as a result of chromatin remodeling). Many features associated with senescent fibroblasts appear to promote conversion to an immunogenic phenotype that facilitates self-elimination by the immune system. Pro-inflammatory cytokines can attract and activate immune cells, the presentation of membrane bound immune ligands allows for specific recognition and promiscuous gene expression may function to generate an array of tissue restricted proteins that could subsequently be processed into peptides for presentation via MHC molecules. However, the phenotypes of senescent cells from different tissues and species are often assumed to be broadly similar to those seen in senescent human fibroblasts, but the data show a more complex picture in which the growth arrest mechanism, tissue of origin and species can all radically modulate this basic pattern. Furthermore, well-established triggers of cell senescence are often associated with a DNA damage response (DDR), but this may not be a universal feature of senescent cells. As such, we discuss the role of DNA damage in regulating an immunogenic response in senescent cells, in addition to discussing less established "atypical" senescent states that may occur independent of DNA damage.

From old organisms to new molecules: integrative biology and therapeutic targets in accelerated human ageing.

Understanding the basic biology of human ageing is a key milestone in attempting to ameliorate the deleterious consequences of old age. This is an urgent research priority given the global demographic shift towards an ageing population. Although some molecular pathways that have been proposed to contribute to ageing have been discovered using classical biochemistry and genetics, the complex, polygenic and stochastic nature of ageing is such that the process as a whole is not immediately amenable to biochemical analysis. Thus, attempts have been made to elucidate the causes of monogenic progeroid disorders that recapitulate some, if not all, features of normal ageing in the hope that this may contribute to our understanding of normal human ageing. Two canonical progeroid disorders are Werner's syndrome and Hutchinson-Gilford progeroid syndrome (also known as progeria). Because such disorders are essentially phenocopies of ageing, rather than ageing itself, advances made in understanding their pathogenesis must always be contextualised within theories proposed to help explain how the normal process operates. One such possible ageing mechanism is described by the cell senescence hypothesis of ageing. Here, we discuss this hypothesis and demonstrate that it provides a plausible explanation for many of the ageing phenotypes seen in Werner's syndrome and Hutchinson-Gilford progeriod syndrome. The recent exciting advances made in potential therapies for these two syndromes are also reviewed.

Hydroxyapatite promotes superior keratocyte adhesion and proliferation in comparison with current keratoprosthesis skirt materials.

AIM: Published clinical series suggest the osteoodontokeratoprosthesis (OOKP) may have a lower extrusion rate than current synthetic keratoprostheses. The OOKP is anchored in the eye wall by autologous tooth. The authors' aim was to compare adhesion, proliferation, and morphology for telomerase transformed keratocytes seeded on calcium hydroxyapatite (the principal mineral constituent of tooth) and materials used in the anchoring elements of commercially available synthetic keratoprostheses. METHODS: Test materials were hydroxyapatite, polytetrafluoroethylene (PTFE), polyhydroxyethyl methacrylate (HEMA), and glass (control). Cell adhesion and viability were quantified at 4 hours, 24 hours, and 1 week using a calcein-AM/EthD-1 viability/cytotoxicity assay. Focal contact expression and cytoskeletal organisation were studied at 24 hours by confocal microscopy with immunoflourescent labelling. Further studies of cell morphology were performed using light and scanning electron microscopy. RESULTS: Live cell counts were significantly greater on hydroxyapatite surfaces at each time point (p<0.04). Dead cell counts were significantly higher for PTFE at 7 days (p<0.002). ss(1) integrin expression was highest on hydroxyapatite. Adhesion structures were well expressed in flat, spread out keratocytes on both HA and glass. Keratocytes tended to be thinner and spindle shaped on PTFE. The relatively few keratocytes visible on HEMA test surfaces were rounded and poorly adherent. CONCLUSIONS: Keratocyte adhesion, spreading, and viability on hydroxyapatite test surfaces is superior to that seen on PTFE and HEMA. Improving the initial cell adhesion environment in the skirt element of keratoprostheses may enhance tissue integration and reduce device failure rates.

Molecular cytogenetic insights into the ageing syndrome Hutchinson-Gilford Progeria (HGPS).

Hutchinson-Gilford Progeria Syndrome (HGPS) is an extremely rare genetic disorder characterized by premature ageing in childhood and serves as a valuable model for the human ageing process in general. Most recently, point mutations in the lamin A (LMNA) gene on chromosome 1q have been associated with the disease, however how these mutations relate to the complex phenotype of HGPS remains to be established. It has been shown that fibroblasts from HGPS patients are frequently resistant to immortalization with telomerase (hTERT), consistent with the idea that the loss of a dominant acting HGPS gene is a pre-requisite for immortalization. In this study we report the first detailed cytogenetic analysis of hTERT-immortalised HGPS cell lines from three patients and one corresponding primary fibroblast culture. Our results provide evidence for a cytogenetic mosaicism in HGPS with a distinctive pattern of chromosome aberrations in all the HGP clones. Chromosome 11 alterations were observed at a high frequency in each immortalised HGPS cell line but were also present at a lower frequency in the corresponding primary cells. Moreover, we were able to identify the 11q13-->q23 region as a potential site of breakage. Our results are therefore consistent with a role of chromosome 11 alterations in the escape from senescence observed in HGPS cells. In addition to this defined rearrangement, we consistently observed complex chromosomal rearrangements, suggesting that HGPS displays features of chromosomal instability.

Camptothecin sensitivity in Werner syndrome fibroblasts as assessed by the COMET technique.

Werner syndrome (WS) is an inherited genetic disease in which individuals display the premature aging of a selected subset of tissues. The disorder results from the loss of function mutations in the wrn gene. Wrn codes for a member of the RecQ helicase family with a unique nuclease domain. There is significant evidence that the role of wrn is to assist in the repair and reinitiation of DNA replication forks that have stalled. Loss of the wrn helicase imposes a distinct set of phenotypes at the cellular level. These include premature replicative senescence (in a subset of cell types), chromosomal instability, a distinct mutator phenotype, and hypersensitivity to a limited number of DNA damaging agents. Unfortunately, most of these phenotypes are not suitable for the rapid assessment of loss of function of the wrn gene product. However, WS cells have been reported to show abnormal sensitivity to the drug camptothecin (an inhibitor of topoisomerase type I). A rapid assay for this sensitivity would be a useful marker of loss of wrn function. The COMET (single-cell gel electrophoresis) assay is a rapid, sensitive, versatile, and robust technique for the quantitative assessment of DNA damage in eukaryotic cells. Using this assay, we have found that a significantly increased level of strand breaks can be demonstrated in WS cells treated with camptothecin compared with normal controls.

A model for the phenotypic presentation of Werner's syndrome.

Werner's syndrome (WS) is a valuable model of accelerated ageing and results from mutations in a recQ helicase (wrn). WS fibroblasts show a mutator phenotype, replication fork stalling, increased rates of mean telomeric loss and accelerated cellular senescence. Senescence has been proposed as a candidate mechanism for the ageing of mitotic tissue. However, some mitotic tissues (such as the immune system) seem unaffected in WS. Is this evidence against a role for cell senescence in ageing? Two experiments resolve this paradox (i) the demonstration that the abbreviated replicative lifespan of WS fibroblasts can be corrected by the ectopic expression of telomerase and (ii) the demonstration that T cells derived from WS patients have the mutator phenotype characteristic of the disease but show no reduction in replicative potential. Since T cells can upregulate telomerase naturally these findings are consistent with a model in which the only wrn-mediated deletions that have a significant effect on replicative lifespan are those at or near the telomere. These data are thus supportive of a role for senescence in the ageing of the immune system. Emerging data on divisional counting mechanisms have the potential to produce many other apparent WS "paradoxes". Accordingly, we propose a general model for the phenotypic presentation of WS, which includes a modification of the Olovnikov model of telomere erosion. Somewhat unexpectedly, this predicts that accelerated senescence should not be observed in all telomerase-negative WS cell types.

Ageing and the nervous system: insights from studies on invertebrates.

Ageing can have profound effects on the post-mitotic organ of behaviour, the brain. As yet the precise causes of these deleterious effects are unknown. However, clear insights into the putative mechanisms and consequences of ageing in the CNS have been achieved through the use of invertebrate models. It is now clear that ageing alters the endogenous properties of neurones, their morphology, the efficacy of the connections that the neurones make with their targets and may even lead to neurone loss. While the precise mechanisms underlying these changes are presently unclear clues from post-mitotic organisms such as C. elegans have provided putative targets which are currently being investigated. It is clear to date that the age-induced changes in CNS function observed in invertebrates are conserved in mammalian species and that further work on invertebrates may provide informative insights in to the mechanisms of neuronal ageing.

Ocular biomaterials and implants.

The maintenance of vision is a key determinant of healthy ageing. This has been facilitated over recent decades by the development of a wide range of implants and biomedical devices to correct the functional deficiencies of disease, age and ocular trauma. This brief overview provides an insight into the structure of this unique organ, the major physiological functions of the component tissues and the present state of the art with respect to modern ocular implants. The review focuses primarily on the existing limitations of existing ocular biomaterials used in the fabrication of contact lenses, intraocular lenses, glaucoma filtration implants, keratoprostheses, intracorneal implants, scleral buckles and viscoelastic replacement agents. The challenge of improving ocular compatibility and ensuring the longevity of indwelling ocular devices is addressed along with the need to improve the physicochemical and mechanical properties of existing ocular biomaterials.

Does MMP-2 expression and secretion change with increasing serial passage of keratocytes in culture?

The effects of ageing on matrix metalloprotease degradation of the extracellular matrix during corneal wound healing are largely unknown. The following study used an in vitro model of ageing to assess changes in MMP-2 RNA expression and protein secretion. Early passage (EP) EK1.BR keratocyte cultures from 14 to 18 cumulative population doublings (cpds) and late passage (LP) cultures from 40 to 47 cpds were used to isolate protein and mRNA samples. Total protein from EP and LP cultures was measured using the Bradford protein assay. Zymographic analysis of EP and LP samples was carried out to compare MMP-2 activity. Northern blot analysis was used to assess changes in MMP-2 mRNA expression by EP and LP cultures, using a digoxigenin (DIG) based chemiluminescent detection system. LP cultures secreted more total protein per cell. MMP-2 but not MMP-9 activity was detected in keratocyte cultures. Densitometric analysis of zymograms and calculation of MMP-2 activity indicated a significant increase in MMP-2 activity per cell (P<0.05, n=11). No difference was observed in the levels of MMP-2 mRNA expressed by EP and LP cultures. An increase in MMP-2 activity per cell by LP cultures suggests that senescent keratocytes increase their degradative capacity. Similar changes in the keratocyte phenotype within the ageing cornea may alter the balanced response necessary for adequate wound healing and may have implications for the therapeutic use of MMP inhibitors in the eye.

Human keratocyte migration into collagen gels declines with in vitro ageing.

Although senescence in various cell types has been shown to have detrimental effects on wound repair, the effect of this phenomenon on corneal function with increasing age has yet to be elucidated. This study investigated the effect of in vitro ageing on keratocyte migration into a collagen gel matrix. The keratocyte cell strain EK1. BR was cultured to late passage and a comparison of early passage migration with that of late passage migration was carried out. Early or late passage keratocytes were seeded onto 6 collagen gels (1.75 mg ml(-1)) for each experiment. Gels were incubated at 37 degrees C for 72 h, stained with calcein AM (0.5 mg ml(-1)) and assayed for cell migration using fluorescent microscopy. Changes in the effect of EGF on keratocyte migration with age were assessed by the addition of EGF (20 ng ml(-1)) to 3 of the 6 gels in each experiment. Proliferative lifespan was measured by immunocytochemical detection of Ki67 activity. This study shows for the first time that keratocyte migration, and migration in response to EGF stimulation, significantly declines with increasing age of keratocytes in culture (P<0.001). As keratocyte migration in response to cytokine stimulation is vital for corneal repair, the accumulation of senescent keratocytes with age may impair corneal wound healing.

A cell kinetic analysis of human umbilical vein endothelial cells.

Cultures of normal human cells 'age' and become senescent in vitro due to a continuously declining mitotic fraction. Although endothelial cells represent a tissue of major relevance in the development of age-related vascular disease, the rate at which these cells senesce has never been systematically measured in culture. Accordingly the population kinetics of human vascular endothelial cells (HUVECs) serially passaged in vitro has been studied in order to determine (i) the rate of decline in the growth fraction; (ii) the rate of increase of the senescent fraction and (iii) the relationship between changes in these parameters and the baseline rate of apoptosis. Immunocytochemical visualisation of the growth fraction using antisera to the proliferation marker pKi67 showed a rate of decline in the growth fraction of 4.43+/-0.31% per population doubling. This was not accompanied by any change in cell cycle time as assessed using time lapse video microscopy. The number of senescent cells within the population increased at a rate of 6.47+/-0.3% as assessed by senescence associated beta-galactosidase activity. The baseline rate of apoptosis as measured by TUNEL remained essentially unchanged (0.31+/-0.07%) during this process. These data show (i) that senescence and apoptosis are unrelated processes in HUVEC and (ii) that senescent cells rapidly and progressively accumulate in dividing populations of endothelial cells. The physiological relevance of these observations is discussed.

Novel materials to enhance keratoprosthesis integration.

BACKGROUND: The successful integration of keratoprostheses (KPros) within the cornea depends in part on peripheral host keratocyte adhesion to anchor the implant in place and prevent epithelial downgrowth. The following study incorporated different acrylate co-monomers with poly(hydroxyethyl methacrylate) (p(HEMA)) and measured the suitability of these materials as potential skirt materials in terms of their ability to enhance keratocyte adhesion to p(HEMA). METHODS: p(HEMA) hydrogels incorporating varying amounts of the acrylate co-monomers methacrylic acid (MA), 2-(dimethylamino)ethyl methacrylate (DEM), or phenoxyethyl methacrylate (PEM) were formed by free radical polymerisation. Keratocytes were seeded onto discs of each material and incubated at 37 degrees C for 72 hours. Assays for viable cell adhesion were carried out. A viability/cytotoxicity assay using solutions of calcein-AM (0.5 mM) and ethidium homodimer-1 (EthD-1) (0.5 microM) were used to measure viable and non-viable cell adhesion, respectively. An ATP assay was also used to quantify cell adhesion in terms of the amount of ATP present following lysis of adherent cells. RESULTS: The viability/cytotoxicity assays indicated that the incorporation of 15 mol% of the co-monomer PEM or of 20 mol% DEM increased cell adhesion to p(HEMA) by at least four times. The ATP assays confirmed the results for PEM but absorption of ATP to the DEM containing hydrogel indicated that the assay was not a suitable measure of cell adhesion to this material. CONCLUSIONS: The properties of p(HEMA) may be moderated to enhance keratocyte adhesion by the incorporation of PEM or DEM suggesting that these may be suitable materials for use in the further development of a novel KPro skirt material.

Cell senescence and human aging: where's the link?

Normal human cells will not divide forever in culture. After a defined number of passages, every culture enters a viable non-dividing state termed senescence. This led to the proposal that the progressive accumulation of senescent cells contributes to (but does not exclusively cause) the aging process. Data now suggest that cell senescence, like apoptosis, occurs as an anti-cancer mechanism. In vivo, extremely low rates of division occur over very long periods of time. Thus, by the end of its lifespan, an organism has performed a surprisingly large amount of cell turnover. In a normal culture the fraction of senescent cells increases smoothly with proliferation in vitro. This process can be accurately modelled by the progressive loss of telomeric sequence (proposed as a counting mechanism for senescence). Telomere length is usually maintained by the enzyme telomerase (repressed in many human tissues). Re-introduction of telomerase prevents the onset of senescence. However, data suggest that a separate telomere-independent pathway exists. Evidence that this pathway is present in humans is now emerging.

Telomerase and the cellular lifespan: implications of the aging process.

The aging process has multiple causes. However, there is now substantial evidence consistent with the hypothesis that (i) all normal mammalian somatic cells have a finite capacity to replicate and (ii) that gradual cell turnover throughout the lifespan of a mammal eventually exhausts this finite capacity. This results in a gradual accumulation of senescent (irreversibly post-mitotic) cells with increasing age. These cells display a radically different phenotype to their growing counterparts, which has the potential to compromise tissue function. Perhaps the best evidence for this is seen in Werner's syndrome, a rare genetic disease, in which patients display most of the features of accelerated aging, together with a profoundly compromised replicative lifespan in certain tissue lineages. Several classes of human cells are now known to count divisions by monitoring the progressive attrition of chromosomal ends (telomeres), leading to the activation of a p53-p21waf-dependent G1 checkpoint. Ectopic expression of telomerase has been shown to prevent senescence in several cell types and offers the potential for interventions in the aging process based on tissue engineering, gene therapy or homeografts. However, this telomere-driven senescence mechanism seems to be absent from rodents, which use telomere-independent means (perhaps based upon p14arf) to count divisions. Similar senescence pathways are now being reported in humans, and this, coupled with the demonstration of tissue-specific telomeric loss rates, has the potential to render strategies based on the use of telomerase dependent on the characteristics of the target tissue. Werner's syndrome may provide strong clues regarding the potential limitations and prospects of such future treatments.

Werner's syndrome T lymphocytes display a normal in vitro life-span.

Werner's syndrome (WS) is an autosomal recessive disorder displaying many features consistent with accelerated ageing. Fibroblasts from WS patients show a distinct mutator phenotype (characterised by the production of large chromosomal deletions) and a profound reduction in proliferative capacity. The disorder results from a mutation in a novel ReqQ helicase. Recently, we demonstrated that the proliferative defect was corrected by the ectopic expression of telomerase. From these data, we propose that mutations in the wrn gene lead to deletions at or near the telomere which reduce the cells replicative life-span. This hypothesis predicts that cell types which retain the ability to upregulate telomerase as part of their response to a proliferative stimulus would fail to show any significant effect of wrn gene mutations upon life-span. Human T lymphocytes represent a well-characterised example of such a cell type. To test the hypothesis, WS T lymphocytes were cultured until they reached replicative senescence. These cultures displayed life-spans which did not differ significantly from those of normal controls. These findings are consistent with the hypothesis that the effects of wrn mutations on replicative life-span are telomere-mediated.

Assessing the in vitro cell based ocular compatibility of contact lens materials.

A series of in vitro assays for determining the biocompatibility of ocular biomaterials have been developed and used to assess the differences in performance of omafilcon A, etafilcon A and nelfilcon A contact lens materials. The assays assessed bacterial attachment, macrophage adhesion, granulocyte adhesion and activation, epithelial cell adhesion and corneal cell contact damage. Overall, omafilcon A was found to be more biocompatible than the other materials although there was no significant difference between the epithelial cell adhesion and granulocyte adhesion and activation on any of the hydrogels. Etailcon A performed less well compared to nelfilcon A and omafilcon A with respect macrophage adhesion and bacterial adhesion. The results indicate that these biological assays can be successfully applied for the testing of contact lens materials and may be particularly useful in the in vitro screening of new extended wear contact lens materials where cell adhesion and activation may have a greater influence on clinical performance.