David Kipling, Dawn L. Jones, S. Kaye Smith, Peter J. Giles, Katrin Jennert-Burston, Badr Ibrahim, Angela N.P. Sheerin, Amy J.C. Evans, William Rhys-Willams and Richard G.A. Faragher,
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.
The pharmacokinetic changes observed in elderly patients are well understood (click here) and allowances can be made for them. However, pharmacodynamic’s is much harder to predict as it requires an understanding of the biological changes associated with ageing (many of which may be individual specific). Insight into these processes has mainly been generated by laboratories focused on the molecular mechanism underlying the ageing process. These groups often have a limited understanding of the pharmacology of the elderly. Therefore, research in this area appears not to have progressed beyond cataloguing the observed drug responses in the elderly.
The accumulation of senescent cells in tissues has been linked to ageing and disease and as such could potentially alter the biological response to drugs in the elderly. When a cell becomes senescent, it undergoes a radically altered phenotype (click here). Microarray analysis of primary human lung fibroblasts (IMR-90) and primary skin fibroblasts (Detroit 551) reported that of the 4183 genes analysed, 165 were down-regulated and 191 up-regulated in senescent IMR-90 cells and 154 down-regulated and 76 up-regulated in senescent Detroit 551 cells compared with their growing counterparts (Chen et al 2004). This degree of alteration in the transcriptome is akin to that seen when cells are induced to differentiate (Truckenmiller et al 2001). Essentially, senescent cells should be treated as a completely different cell type from when they were growth competent. Therefore, more research should be carried to determine whether or not senescent cells display an altered responsiveness to pharmacological agents.
By bridging the gap between pharmacokinetic and pharmacodynamic studies and molecular gerontology it is hoped that pharmaceutical intervention might one day be more precisely targeted to the age of the patient (and thus, the biological status of the target tissue). It is anticipated that the development of in-vivo and in-vitro models of tissue ageing will facilitate the necessary advances in pharmacogerontology.
Vascular endothelial senescence: from mechanisms to pathophysiology. Erusalimsky JD. J Appl Physiol. 2009 Jan;106(1):326-32. Epub 2008 Nov 26.
Telomere attrition and accumulation of senescent cells in cultured human endothelial cells. Hastings R, Qureshi M, Verma R, Lacy PS, Williams B. Cell Prolif. 2004 Aug;37(4):317-24
Endothelial Cell Senescence in Human Atherosclerosis. Minamino et al. Circulation. 2002;105:1541.)
A cell kinetic analysis of human umbilical vein endothelial cells. Kalashnik et al. Mech Ageing Dev. 2000 Dec 1;120(1-3):23-32.
Vascular smooth muscle cells
Vascular smooth muscle cells undergo telomere-based senescence in human atherosclerosis: effects of telomerase and oxidative stress. Matthews et al, Circ Res. 2006 Jul 21;99(2):156-64. Epub 2006 Jun 22
Microarray analysis of senescent vascular smooth muscle cells: A link to atherosclerosis and vascular calcification. Burton et al (2009) Experimental gerontology 2009 Oct;44(10):659-65 PubMed ID:(19631729)
Beta-galactosidase histochemistry and telomere loss in senescent retinal pigment epithelial cells. Matsunaga et al, Invest Ophthalmol Vis Sci. 1999 Jan;40(1):197-202
T cell replicative senescence: pleiotropic effects on human aging. Effros RB, Ann N Y Acad Sci. 2004 Jun;1019:123-6
The role of CD8+ T-cell replicative senescence in human aging. Effros RB, Dagarag M, Spaulding C, Man J. Immunol Rev. 2005 Jun;205:147-57
Microglial senescence: does the brain's immune system have an expiration date? Streit WJ Trends Neurosci. 2006 Sep;29(9):506-10. Epub 2006 Jul 20
The role of microglial cellular senescence in the aging and Alzheimer diseased brain. Flanary B, Rejuvenation Res. 2005 Summer;8(2):82-5
Astrocytes aged in vitro show a decreased neuroprotective capacity. Pertusa et al, J Neurochem. 2007 May;101(3):794-805. Epub 2007 Jan 23
Demonstration of cellular aging and senescence in serially passaged long-term cultures of human trabecular osteoblasts. Kassem et al. Osteoporos Int. 1997;7(6):514-24.
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Aging, articular cartilage chondrocyte senescence and osteoarthritis. Martin and Buckwalter, Biogerontology. 2002;3(5):257-64
Pancreatic Beta cells
Pancreatic beta cell senescence contributes to the pathogenesis of type 2 diabetes in high-fat diet-induced diabetic mice. Sone H, Kagawa Y., Diabetologia. 2005 Jan;48(1):58-67. Epub 2004 Dec 29
Role of replicative senescence in the progression of fibrosis in hepatitis C virus (HCV) recurrence after liver transplantation. Trak-Smayra et al, Transplantation. 2004 Jun 15;77(11):1755-60
Hepatocyte telomere shortening and senescence are general markers of human liver cirrhosis. Wiemann et al, FASEB J. 2002 Jul;16(9):935-42
Increased expression of senescence-associated cell cycle inhibitor p16INK4a in deteriorating renal transplants and diseased native kidney. Melk et al, Am J Transplant. 2005 Jun;5(6):1375-82
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Premature senescence of highly proliferative endothelial progenitor cells is induced by tumor necrosis factor-alpha via the p38 mitogen-activated protein kinase pathway. Zhang et al, FASEB J. 2009 May;23(5):1358-65. Epub 2009 Jan 5
Alzheimer’s disease (AD) is the most common form of dementia and is found predominantly in people aged over 65 years. It is progressive, degenerative and currently irreversible. Features of the disease include memory loss, decreased reasoning and judgment and changes in mood, behaviour and personality. The majority of the research into AD is focused on protein plaques (mostly made up of a protein called B-amyloid) and neurofibrillary tangles (composed of a protein called tau) found in the brain of AD patients. The role of amyloid plaques and neurofibrillary tangles on the functioning of the brain is poorly understood and research is ongoing. This article however, neither focuses on plaques or tangles, but instead on the role of cellular senescence (specifically microglial and astrocytes) in the development and/or progression of AD.
Microglial cells and Astrocytes in Alzheimer’s
Both microglial and astrocytes are mitotic cells which have been shown to undergo cellular senescence (Streit, 2006 and Pertusa et al, 2007). Moving around the brain, microglia function as immune cells to remove damaged neurons, plaques and infecting micro-organisms (Pivneva, 2008). Astrocytes appear to have numerous responsibilities including providing nutrients to neurons and neuronal maintenance (Seth and Koul, 2008, Rodriguez et al 2009). Therefore, without microglia and astrocytes, or a decline in their function, there would most likely be an increase in neuronal damage and this could manifest as disease. An accumulation of senescent microglial and astrocytes in the brain could lead to such a functional decline. However, as with a number of cell types, little is known about the senescent-specific phenotype of microglia and astrocytes and little work has been carried out to investigate the possible contribution senescent cells may have on the development/progression of AD.
An interesting study by Streit et al (2007) demonstrated that the presence of amyloid protein promotes cellular senescence in microglial cells. Amyloid protein causes microglial cells to become activated and thus proliferate to facilitate amyloid removal. Constant activation and cell turnover of microglial cells would result in gradual telomere attrition and thus an increased appearance of senescent cells. Apolipoprotein E (apoE) also plays a role in the degradation and clearance of amyloid protein by astrocytes. However, varients of this gene have been shown to be a major risk factor in the development of late onset AD (Wang and Ding, 2008). Research has shown that this varient enhances the production of amyloid protein (Ye et al, 2005), which may consequently lead to an increase in microglial activation and accelerated appearance of senescent microglia. The presence of amyloid is just one example of how microglial cell senescence may become accelerated, but other currently unknown risk factors may also have the same impact.
Little is known about the mechanisms by which astrocytes become activated and it can only be speculated as to the effects senescent astrocytes would have on the brain (if any). Astrocytes have been shown to become activated and proliferate in culture in the presence of cytokines and growth factors (Selmaj et al 1990) and by neuroinflammation in the brain (Norris et al, 2005). General features of a senescent phenotype appear to be an up-regulation of pro-inflammatory cytokines, growth factors and matrix degrading proteins. If this is true for senescent microglial cells, then it could be speculated that the accumulation of senescent microglial cells (if they persist in tissue) may consequently lead to the activation and proliferation of astrocytes. Interestingly, interleukin 1 (a cytokine known to stimulate astrocyte proliferation) is elevated in both Down syndrome (risk factor for AD) and in AD (Griffin et al, 1989). This up-regulation of interleukin 1 appears to orinate from activated microglial (Mrak, 2001). Inflammaotory mediators have long been shown to be up-regulated in pathologically vulnerable regions of the brain in AD (Rogers, 2008). The constant proliferation of astrocytes would eventially result in the accumulated appearance of senescent astrocytes (further research needs to be carried out to determine if astrocyte numbers increase in AD progression). This means that neurons will become functionally impaired, damaged or lossed. Pertusa et al (2007) found that aged astrocytes in vitro show a decreased neuroprotective cacpacity. Long-term cultures of astrocytes demonstrated positive staining for senescence-associated-beta-galactosidase (a senescent marker, Dimri et al, 1995) suggesting that this functional decline is associated with the senescent phenotype of astrocytes.
Neuronal loss may not be a major problem initally since they are most likey replaced by neural stem/progenitor cells (Taupin, 2006). However, these cells have also been shown to undergo cellular senescence or become functionally impaired with age (Sharpless and DePinho 2007, Ruzankina and Brown 2007). This means, the brain would reach a point where neurons are being lost without replacement.
A theoretical consequence of the senescent astrocyte phenotype might be related to that which is observed in senescent vascular endothelial cells (ECs). Senescent vascular ECs show a reduction in nitric oxide (NO) production by eNOS (Minamino et al, 2002). In the vascular system, NO signals the surrounding smooth muscle to relax, thus resulting in vasodilation and increasing blood flow. A reduction in NO would therefore restrict bloodflow and reduce oxygen supply to much needed tissues. NO reduction has been suggested to be a significant risk factor for cardiovascular disease.
Astrocytes perform many functions, including biochemical support of endothelial cells which form the blood-brain barrier. eNOS activity has also been shown to be present in astrocytes (Lin et al, 2007), but the impact of cellular senescence (if any) on eNOS activity on this cell type is currently lacking. However, since it has been shown that astrocytes play a direct role in controlling blood flow in the brain (Koehler et al 2009) and if senescent astrocytes do reduce the synthesis of NO, then this may lead to a reduction of blood flow to certain areas of the brain. This means less oxygen and potential cell death..
One final point of mention. In AD, the hippocampus (important in long-term memory) and the neocortex (higher level cognitive function such as language, learning and memory) are one of the first areas of the brain to undergo damage (Scheff and Price, 2006). Interestingly, both microglial and astrocytes have been shown to specifically proliferate in the hippocampus and temporal neocortex following global cerebral ischemia in young adult monkey brain (Tonchev et al, 2003). This region specific proliferation of microglial cells and astrocytes, the same region affected in AD, provides some link that these two cell types may play a role in the pathogenesis of AD.
To date little work has been carried out to investigate microglial cell and astrocyte senescence in AD. Little is known about the senescent phenotype of microglial cells and astrocytes and what impact (if any) this phenotype may consequently have on the brain. A number of points in this article can only be speculative, but based on what we know about the phenotype of other senescent cell types and the theoretical impact of their presence, it is not difficult to envisage a role for cellular senescence in AD development and/or progression.