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.

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.

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).

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.

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.

A facile, stereoselective, one-pot synthesis of resveratrol derivatives.

BACKGROUND: Compounds based on trans-1,2-diphenylethene are the subject of intense interest both for their optical properties and as potential leads for drug discovery, as a consequence of their anticancer, anti-inflammatory and antioxidant properties. Perhaps the best known of these is trans-3,5,4'-trihydroxystilbene (resveratrol), that has been identified as a promising lead in the search for anti-ageing therapeutics. RESULTS: We report here a new, convenient, one-pot stereo-selective synthesis of resveratrol and other trans-stilbene derivatives. A wide range of known and novel "Resveralogues" were synthesised by using this simple protocol, including examples with electron donating and electron withdrawing substituents, in uniformly high yield. The structures of all compounds were confirmed by standard methods including (1)H and (13)C NMR, IR and High Resolution Mass spectroscopy. CONCLUSIONS: We have established a simple and convenient protocol for resveralogue synthesis. It is readily scalable, and sufficiently robust and simple for ready use in automated synthesis or for library development of resveralogues. This supersedes previously reported synthetic methods that required inert conditions, extensive purification and/or costly reagents. Graphical abstractOne-pot preparation of diverse Resveralogues - high yields of product with minimal purification.

Fibroblast clones from patients with Hutchinson-Gilford progeria can senesce despite the presence of telomerase.

Hutchinson-Gilford progeria (HGP) is a genetic disorder in which individuals prematurely display features of ageing. Mutations in LMNA (lamin A) have recently been shown to underlie HGP, although how such mutations lead to the complex phenotype seen in the disease remains unclear. HGP is often associated with the premature replicative senescence of dermal fibroblasts. Normally dermal fibroblast senescence is initiated by erosion of chromosomal ends (telomeres) resulting from sustained cell division. Since ectopic expression of telomerase reproducibly immortalises human dermal fibroblasts, it is of interest to determine whether HGP fibroblasts immortalise via the same route, and at the same frequency. Three strains of HGP fibroblasts (AGO6917A, AGO6297B and AGO8466) were infected with a retroviral vector expressing the catalytic subunit of telomerase (hTERT). Here we report that fibroblast clones derived from HGP donors frequently fail to immortalise with telomerase. Of the 15 independently isolated clones from the three donors, five failed to immortalise despite the restoration of telomerase activity and the stabilisation of telomere length. In contrast, out of four clones isolated from a culture of hTERT transduced control fibroblasts, no failures to immortalise were detected. This suggests a novel cellular phenotype in HGP, one whereby the HGP mutation confers resistance to 'telomerisation'.

Characterization of cellular senescence mechanisms in human corneal endothelial cells.

The human cornea is a tri-laminar structure composed of several cell types with substantial mitotic potential. Age-related changes in the cornea are associated with declining visual acuity and the onset of overt age-related corneal diseases. Corneal transplantation is commonly used to restore vision in patients with damaged or diseased corneas. However, the supply of donor tissue is limited, and thus there is considerable interest in the development of tissue-engineered alternatives. A major obstacle to these approaches is the short replicative lifespan of primary human corneal endothelial cells (HCEC). Accordingly, a comprehensive investigation of the signalling pathways and mechanisms underpinning proliferative lifespan and senescence in HCEC was undertaken. The effects of exogenous human telomerase reverse transcriptase expression, p53 knockdown, disruption of the pRb pathway by over-expression of CDK4 and reduced oxygen concentration on the lifespan of primary HCEC were evaluated. We provide proof-of-principle that forced expression of telomerase, when combined with either p53 knockdown or CDK4 over-expression, is sufficient to produce immortalized HCEC lines. The resultant cell lines express an HCEC-specific transcriptional fingerprint, and retain expression of the corneal endothelial temperature-sensitive potassium channel, suggesting that significant dedifferentiation does not occur as a result of these modes of immortalization. Exploiting these insights into proliferative lifespan barriers in HCEC will underpin the development of novel strategies for cell-based therapies in the human cornea.

Microarray analysis of senescent vascular smooth muscle cells: A link to atherosclerosis and vascular calcification.

Little is known about the senescent phenotype of human vascular smooth muscle cells (VSMCs) and the potential involvement of senescent VSMCs in age-related vascular disease, such as atherosclerosis. As such, VSMCs were grown and characterised in vitro to generate senescent VSMCs needed for microarray analysis (Affymetrix). Comparative analysis of the transcriptome profiles of early (14 CPD) and late (39-42 CPD) passage VSMCs found a total of 327 probesets called as differentially expressed: 149 are up-regulated in senescence and 178 repressed (p-value<0.5%, minimum effect size of at least 2-fold differential regulation, explore data at http://www.madras.cf.ac.uk/vsmc). Data mining shows a differential regulation of genes at senescence associated with the development of atherosclerosis and vascular calcification. These included genes with roles in inflammation (IL1beta, IL8, ICAM1, TNFAP3, ESM1 and CCL2), tissue remodelling (VEGF, VEGFbeta, ADM and MMP14) and vascular calcification (MGP, BMP2, SPP1, OPG and DCN). The microarray data for IL1beta, IL8 and MGP were validated by either, ELISA, Western blot analysis or RT-PCR. These data thus provide the first evidence for a role of VSMC senescence in the development of vascular calcification and provides further support for the involvement of senescent VSMCs in the progression of atherosclerosis.