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BASIC SCIENCE REVIEW

Synopsis on cellular and

Joseph D. Raffetto, MD,a Martin Leverkus, MD,b Hee-Young Park, PhD,a and James O. Menzoian, MD,a,c Boston, Mass, and Wuerzburg, Germany

In this review of basic vascular science, the two cellu- characterization of diploid human derived from lar events consisting of cellular senescence and apoptosis fetal tissues. Their study results demonstrated unequivo- are defined, and their molecular properties are elucidated. cally that cells in culture had a finite number of subculti- We emphasize that these are two unique cellular functions vations (also referred to as passage) of approximately and should not be used interchangeably. Their importance 50 before achieving growth arrest. These findings were in vascular biology is only recently beginning to be real- not caused by medium components or culture conditions, ized, and they may have potential mechanistic implications but instead were an inherent property of the cells them- in providing a better understanding of arterial and venous selves in culture. This phenomenon was interpreted as diseases. Cellular senescence has been shown to be a char- aging at the cell level, therefore refuting the concept of acteristic in venous ulcer grown in culture and a cell immortality, and was established as an in vitro model marker of endothelial cells found in diseased arteries. for which exhaustion of the doubling capacity in human Likewise, apoptosis is responsible for cell turnover and cel- diploid fibroblasts is recognized as cellular senescence. In lular environment homeostasis and appears to be altered in the last 35 years, many cell types from various animal diseased arterial and venous tissues, and it is possibly a species have been studied and shown to have a finite contributing factor in the pathophysiology of atheroscle- replicative life span. By definition, postmitotic cells, which rosis. In addition, the difference between apoptosis and do not replicate, do not undergo cellular senescence. cell will be highlighted. Instead, cellular senescence affects the of divid- ing cells only. CELLUAR SENESCENCE In the last decade, the model of cellular senescence has The hallmark of cellular or replicative senescence is an been applied in vitro to human cells comprising living tis- irreversible arrest of with maintenance of sues, with the implication of possibly having an important cell functions. At the turn of the 20th century, Alexis role in the function of tissue integrity. The role of cellular Carrel, a distinguished Nobel laureate, surgeon, and cell senescence has been extended to explain the aging culturist, demonstrated that fibroblasts from the chick process, tumor suppression, and impaired tissue function heart were immortal in tissue culture. Because of this find- resulting in poor wound healing. In the aforementioned ing and the duplication of these results by other laborato- normal process of aging and disease states related to neo- ries, the prevailing dogma until the 1960s was the belief plastic transformation and wound impairment, it is worth- that all cultured cells were immortal. Because it was rec- while to review the expected replicative capacity of cells as ognized that on occasion and at exceedingly low frequen- they undergo sequential population doublings. As seen in cies immortal cells can arise spontaneously from normal Fig 1, the proliferative capacity of the cells decreases with cell cultures and that external stimuli such as carcinogens, increasing population doublings. As fibroblasts approach radiation, and oncogenic viruses are required for cell senescence, each cell at a given passage will have under- transformation and immortality, the hypothesis of Carrel gone a finite number of divisions, and the population of was challenged. In 1961, the extensive tissue culture stud- fibroblasts as a whole will be at varying stages toward ies of Hayflick and Moorhead described the isolation and senescence, depending on the number of population dou- blings. The entire population of fibroblasts can be said to From Boston University Medical Center,a University Hospital of have reached cellular senescence when every cell in the Wuerzburg, Germany,b and Boston University School of Medicine.c culture has completed its replicative life span. At this stage, Competition of interest: nil. J Vasc Surg 2001;34:173-7. growth arrest is irreversible, and a senescent cell cannot be Reprint requests: James O. Menzoian, MD, Boston Medical Center, stimulated by any physiologic mitogens to initiate DNA Department of Surgery, Section of Vascular Surgery, D506, One Boston replication. Medical Center Pl, Boston, MA 02118. Copyright © 2001 by The Society for Vascular Surgery and The American MOLECULAR CHARACTERISTICS Association for Vascular Surgery. OF CELLULAR SENESCENCE 0741-5214/2001/$35.00 + 0 24/9/115964 doi:10.1067/mva.2001.115964 The hallmark of cellular senescence is the irreversible 173 JOURNAL OF VASCULAR SURGERY 174 Raffetto et al July 2001

receptor structural moiety and in the postreceptor sec- ondary messenger pathways. The changes in secondary messenger signal transduction involve different molecules consisting of phospholipid metabolite arachidonic acid and the production of prostaglandins, diacylglycerol, pro- tein kinase A and C, cyclic adenosine monophosphate, and intracellular calcium. There are a number of normally expressed genes in the that are not expressed in senescent cells. These genes, such as cyclin A, proliferating cell nuclear antigen, thymidine kinase, and DNA polymerase α, are regulated by the E2F. The inability of senescent cells to synthesize and replicate DNA during the cell cycle Fig 1. Replicative capacity of normal somatic mammalian cells involves in part the suppression of at least three positive- undergoing sequential population doublings (PD; 1PD = popula- acting genes. In senescent fibroblasts, the expression of tion of cells that has doubled in number). Replicative capacity c-fos proto-, helix-loop-helix id-1 and id-2 depends on type, age, environmental influences (eg, viruses), and pathologic state of cells. During normal proliferative capacity (eg, genes, and the components of the E2F transcriptional newborn fibroblasts, adult dermal fibroblasts), cells undergo factor is inhibited, triggering the inability of the cell for many population doublings before replicative exhaustion (A). DNA synthesis. The alterations of the growth-regulating Fibroblasts cultured from tissue with concomitant pathology (eg, genes in senescent fibroblasts are intimately coupled to venous ulcers) have reduced growth potential and are at a higher various cell-cycle dependent proteins. The expression of stage toward reaching cellular senescence than normal fibroblast cell-cycle regulator proteins tumor suppressor , cyclin- (B). At the end of life span of cell, proliferative capacity has been dependent kinase inhibitor (cip1/sdi1/waf1) and exhausted, and cells are senescent (C). Cells that have completed (cdkn2/ink4a), and the tumor suppressor retinoblas- their PDs can escape cellular senescence and become immortal toma susceptibility protein pRb is involved directly or either spontaneously (rare) or be transformed by viruses to con- indirectly in the regulation of cell proliferation. The over- tinue to replicate and extend their normal PDs (D). expression of p21 is an important regulator of cell prolif- eration in senescent cells. Because p21 (and also p16) inhibits cyclin-dependent protein kinases, which leads to the constitutive underphosphorylation of pRb and E2F growth arrest of the cell, with preservation of metabolic suppression, this protein plays a major role in inhibiting function. Morphologically, senescent cells are larger in size cell proliferation in senescent cells. It is suggested by evi- and can have polygonal shapes. This appearance may be dence that inactivation of p21 by genetic modulation can caused in part by a number of growth regulatory depen- bypass the events of senescence in human diploid fibro- dent genes being repressed. Although these aspects are the blasts and that oncogenic ras (a proliferation cell-regulat- primary characteristics of a senescent cell, the molecular ing protein) can lead to accumulation of p16 and p53, events involving alterations in signal transduction and the leading to premature cellular senescence. regulation of DNA replication are fundamental in distin- Senescent cells display altered differentiated functions, guishing senescent cells from replicating cells and cells which can impart changes on the extracellular environ- undergoing apoptosis. Fig 2 is a schematic representation ment. Senescent cells display resistance to apoptosis prob- of a senescent cell, demonstrating the effect of physiologic ably through the overexpression of Bcl-2 protein. Because mitogens or environmental stimuli on the molecular path- senescent cells would resist programmed cell death, invari- ways involving the interaction of cell receptors, secondary ably this would lead to the accumulation of these cells messenger moieties, and regulatory proteins leading to the within tissues. The accumulation of senescent cells in vivo inhibition of DNA replication. The loss of proliferative that have achieved growth arrest has been postulated to capacity in senescent cells is partially explained by the pro- have consequences affecting aging, neoplastic differentia- found attenuated response to growth factors, such as basic tion, and impaired tissue integrity and healing. A recent fibroblast , epidermal growth factor, trans- senescent marker called senescent-associated β galactosi- forming growth factor, and platelet-derived growth factor. dase (SA β-gal) persists in aged tissues and increases with Studies in normal human diploid fibroblasts from the WI- increasing population doublings. SA β-gal staining of cul- 38 cell line have demonstrated that the -receptor tured human fibroblasts, which is a marker for cellular binding, receptor density (number of receptors per cell senescence, results in a perinuclear blue staining that char- surface area), and the affinity of the receptor for the ligand acterizes these fibroblasts as senescent. Further changes in are unchanged in early passage (presenescent) versus late senescent cells involve the overproduction of matrix me- passage (senescent) fibroblasts. The mechanism for the talloproteinases, collagenases, and gelatinases and de- attenuated response of the cell receptor to the mitogenic creased amounts of metalloproteinase inhibitors. The stimuli appears to be a function of both alterations in the imbalance of elevated proteinase activity compared with JOURNAL OF VASCULAR SURGERY Volume 34, Number 1 Raffetto et al 175

Fig 2. Signaling pathways leading to cellular senescence and DNA inhibition. Various ligands, hormones, and (M2 , M3) bind to cell surface receptors (R2 , R3), or growth factors (M1) bind to specific growth factor receptor (GF R1). Signal transduction to initiate DNA replication involves various mem- brane-associated proteins, adenylate cyclase (AC), tyrosine protein kinase (TPK), G protein (GP), phos- pholipase C (PLC), and protein kinase C (PKC). Phosphotidylinositol-4,5-biphosphate (PIP2 ) is the substrate for PLC to form secondary messengers inositol triphosphate (IP3) and diacylglycerol (DAG). In turn, DAG is important in activating PKC by membrane translocation. Senescent cells have attenuated response to signal transduction (double bars), leading to inhibition of DNA synthesis. Arrest of DNA syn- thesis is also a result of increased metabolites of phospholipids (PL) by action of phospholipase A2 (PLA2) to produce elevated levels of arachidonic acid (AA) and prostaglandin E2 (PGE2) and by inhibition of cyclin-dependent protein kinases (CDK) by overexpression of p21 and p16, causing underphosphoryla- tion of pRb (ie, decrease pRb-PO4) and consequently inhibition of necessary for DNA replication. DHFR, Dihydrofolate reductase; TK, thymidine kinase; DNA poly α, DNA polymerase alpha; PCNA, cofactor proliferating cell nuclear antigen.

antiproteinase has been implicated not only in the degra- reperfusion injury of ischemic tissue and in brain and dation of important extracellular structural proteins like myocardial tissues at risk from further insult during a fibronectin, but also in degrading growth factors and their stroke and myocardial infarction, respectively. receptors. It appears that the accumulation of senescent Apoptosis should be distinguished from necrosis. cells in tissues can have profound effects on neighboring Necrotic cell death is pathologic and results from acute cells and ultimately on the normal aging process and in cellular injury, leading to rapid cellular edema and lysis. In disease states. contrast, cell death by apoptosis has the hallmark of con- trolled autodigestion of the cell. This process leads to the APOPTOSIS formation of apoptotic bodies within the intact cell plasma Apoptosis, a word derived from the Greek word membrane. Unlike cellular necrosis, which is accompanied “falling leaves,” is defined as a programmed cell death. by an inflammatory response because of the loss of mem- Apoptosis has recently received a lot of attention in medi- brane integrity, an important feature of apoptosis is that cine and science because of its importance during organ- elimination of apoptotic cells by mononuclear cell phago- ism development, pathogenesis, and cell transformation. cytosis occurs without the induction of an inflammatory Apoptosis is a fundamental biologic process required to response. Apoptosis occurs when cells commit suicide in maintain the integrity and homeostasis of a multicellular response to exogenous stimuli or intrinsic signals to main- organism. The etiology of many intractable human dis- tain homeostasis of the organism. The events that commit eases, including neurodegenerative disorders such as a cell to either a path of apoptosis or necrotic cell death Alzheimer’s and Parkinson’s diseases and , is associ- after a specific stimulus are dictated in the former and not ated with alterations in cell apoptosis. Furthermore, apop- the latter by the activation of the central cell death signal tosis is implicated in the progression of cell death during either through a specific set of surface death receptors or JOURNAL OF VASCULAR SURGERY 176 Raffetto et al July 2001

Fig 3. Cellular events during apoptosis and necrosis. Extrinsic or intrinsic signals initiate a cascade of events that will cause cell to undergo either apoptosis (specific DNA fragmentation) or necrosis (nonspe- cific DNA degradation). Apoptosis involves activation of specific death receptors (R1, R2, and R3) or direct DNA damage. A central death signal initiates cell death effector machinery, with activation of caspases with DNA fragmentation ensuing. Bcl-2 protein blocks activation of caspases. Necrosis path of cell death involves reduction of adenosine triphosphate (ATP) generation, leading to cellular edema from inhibition of sodium-potassium adenosine triphosphatase (ATPase) pump (ATPase Na/K) and DNA degradation and cell membrane rupture.

directly within the cell by drugs, toxins, or radiation (Fig These proteases are called caspases (cysteinyl aspartate- 3). The signals for apoptosis induction can be intrinsic or specific proteinases), which are inactive precursor polypep- extrinsic. There are many inducers of apoptosis, such as tides, and in turn are cleaved at specific aspartate residues physiologic activators (tumor necrosis factor [TNF], neu- and assembled into active heterotetramer proteases that rotransmitters, growth factor withdrawal), oxygen reactive ultimately begin to digest DNA in a defined and specific metabolites, viral infection, chemotherapeutic drugs, radi- fashion. One of the signature changes associated with ation (UV and gamma), and toxins. These stimuli can apoptosis is DNA fragmentation. Whereas necrosis- direct their effect by activating specific death receptors, induced death causes nonspecific degradation of chromo- causing perturbations of the cell membrane or metabolic somal material, in programmed cell death specific sites of functions, or by causing direct DNA damage. Once a cell DNA are rapidly restricted or cut such that it creates pre- has been induced to go through apoptosis, a cascade of dictable DNA fragments measuring in multiples of 180 events occurs. The first event is provocation of the apop- base pairs. In human physiology, apoptosis plays a very totic response. For activation of the cell surface receptors, important role. Cells that are constantly exposed to UV ligands, which are structurally related to the TNF gene radiation, such as cells, or cells that have a limited life superfamily (with the exception of nerve growth factor), span tend to be resistant to apoptosis. Resistance to apop- bind and stimulate a complex family of death receptors tosis for these cells is developed for long-term survival. In that also belong to the TNF gene superfamily. These contrast, immune cells are very susceptible to apoptosis. death receptors share a similarity in possessing a cysteine- This is a physiologic attempt to regulate autoantibody rich extracellular domain and a cytoplasmic homologous production. Pathogenesis occurs when a cell type loses its sequence named the death domain. There are a number of original susceptibility to apoptosis. When immune cells are death receptors that have been characterized, such as TNF activated, they produce antibodies against foreign pro- receptor family, nerve growth factor receptor, CD95 teins; however, as soon as antibodies are produced, apop- receptor (also called Fas or Apo1), death receptor 3 (also tosis is induced, so that cells producing particular called DR3, Apo3, LARD, or TRAMP), and DR5 (also antibodies are quickly destroyed. Apoptosis associated called Apo2, TRAIL-R2, TRICK 2, or KILLER). After with the vascular diseases is not well known, and this is an the activation of the death receptors by the specific stim- area of research that could be further explored. uli, a central cell death signal is transduced, triggering As described, the hallmark of cellular senescence is the apoptosis by activation of a group of cysteine proteases. loss of proliferative capacity, whereas the hallmark of apop- JOURNAL OF VASCULAR SURGERY Volume 34, Number 1 Raffetto et al 177

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