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Biomedical subjects

R G Faragher

Publications and source records attributed to R G Faragher.

At least 19 recordsLinked to original sources
PubMed2001

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.

Cell Division↗
PubMed2001

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.

Animals↗
PubMed2001

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.

Aging↗
PubMed2000

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.

Cells, Cultured↗
PubMed2000

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.

Apoptosis↗
PubMed2000

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.

Cell Line↗
PubMed2000

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.

Journal Article↗
PubMed2000

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.

Aging↗
PubMed2000

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.

Adenosine Triphosphate↗
PubMed2000

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.

Aging↗
PubMed1999

A standard strain of human ocular keratocytes.

The ability of an injured cornea to regenerate from deep tissue trauma is largely due to wound healing processes mediated by the surviving stromal keratocytes. Despite the importance of the wound healing process, and the ease with which keratocytes can be grown in tissue culture, a standardised strain of the cells has never been made available. Accordingly, this study reports a strain of human embryonic keratocytes, designated EK1.BR as a research tool for the ophthalmic community. EK1.BR has been characterised with respect to life-span, fraction of dividing cells and maintenance of a keratocyte phenotype in culture. It is hoped that these cells will prove useful in the in vitro study of stromal wound healing and the characterisation of keratocyte gene expression.

Aged↗
PubMed1998

How might replicative senescence contribute to human ageing?

Cell senescence is the limited ability of primary human cells to divide when cultured in vitro. This eventual cessation of division is accompanied by a specific set of changes in cell physiology, morphology, and gene expression. Such changes in phenotype have the potential to contribute to human ageing and age-related diseases. Until now, senescence has largely been studied as an in vitro phenomenon, but recent data have for the first time directly demonstrated the presence of senescent cells in aged human tissues. Although a direct causal link between the ageing of whole organisms and the senescence of cells in culture remains elusive, a large body of data is consistent with cell senescence contributing to a variety of pathological changes seen in the aged. This review considers the in vitro phenotype of cellular senescence and speculates on the various possible routes whereby the presence of senescent cells in old bodies may affect different tissue systems.

Aging↗
PubMed1997

Different kinetics of senescence in human fibroblasts and peritoneal mesothelial cells.

Senescence has been reported for a wide variety of human cell types. In cultures of human fibroblasts the process is due to a percentage of the cells becoming senescent at each passage rather than all the cells entering senescence simultaneously at the end of the life span. By measuring the percentage of fibroblasts which are still cycling at each passage, a rate of decline in the growth fraction, which mirrors the rate of senescence, can be obtained. However, such an analysis has never been undertaken in multiple cell types using the same method to identify cycling cells. It is thus unknown if the rate of senescence is the same or different in cultures of different human cell types. To answer this question the rates of decline in the cycling fractions were simultaneously measured in two cultures of human cells (AGO7086A, peritoneal mesothelial cells; and 2DD, human dermal fibroblasts) which have practically identical in vitro life spans. 2DD fibroblasts showed a rate of decline of 0.89% cycling cells per population doubling when the data obtained were fitted to a simple linear equation. However, AGO7086A gave a decline of approximately 2.2% per population doubling. Thus mesothelial cells enter senescence significantly faster than fibroblasts (P < 0.001). This decline in the growth fraction was accompanied by an increasing fraction of mesothelial cells which retained detectable endogenous beta-galactosidase activity at pH 6. Such activity has previously been shown to be associated with senescent human fibroblasts. These findings suggest that the process of senescence has common features in different cell lineages but that the rate of the process can differ markedly between them.

Biomarkers↗
PubMed1997

Cycling Werner's syndrome fibroblasts display calcium-dependent potassium currents.

Werner's Syndrome (WS) fibroblasts undergo premature senescence. Two hypotheses have been proposed to explain this phenomenon: (i) the phenotype is due to the overexpression of senescence-specific proteins in every cell in the population. Such proteins are known to suppress calcium-dependent potassium currents. (ii) The WS mutation greatly increases the proportion of cells that stop cycling at each generation and become senescent. If hypothesis (i) is correct, such currents should be suppressed in all WS fibroblasts; whereas hypothesis (ii) predicts that they will be retained in the cycling fraction of the population. To distinguish between these hypotheses whole-cell patch-clamp currents were recorded from cycling cells. Slowly activating outward calcium-dependent potassium currents were detected in both cycling WS and control fibroblasts. These findings support hypothesis (ii): the premature senescence of WS fibroblasts is due to an increased rate of transition from cycling to senescence in the total cell population.

Calcium↗