Review

Hallmarks of senescence and aging

Slavica Dodig*1, Ivana Čepelak1, Ivan Pavić2
1Department of Medical Biochemistry and Hematology, Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia
2Department of Pulmonology, Allergology and Immunology, Children’s Hospital Zagreb; School of Medicine, University of Zagreb,
Zagreb, Croatia

*Corresponding author: slavica.dodig@zg.t-com.hr

Abstract
The complex process of biological aging, as an intrinsic feature of living beings, is the result of genetic and, to a greater extent, environmental factors
and time. For many of the changes taking place in the body during aging, three factors are important: inflammation, immune aging and senescence
(cellular aging, biological aging). Senescence is an irreversible form of long-term cell-cycle arrest, caused by excessive intracellular or extracellular
stress or damage. The purpose of this cell-cycles arrest is to limit the proliferation of damaged cells, to eliminate accumulated harmful factors and to
disable potential malignant cell transformation. As the biological age does not have to be in accordance with the chronological age, it is important to
find specific hallmarks and biomarkers that could objectively determine the rate of age of a person. These biomarkers might be a valuable measure
of physiological, i.e. biological age. Biomarkers should meet several criteria. For example, they have to predict the rate of aging, monitor a basic pro-
cess that underlies the aging process, be able to be tested repeatedly without harming the person. In addition, biomarkers have to be indicators of
biological processes, pathogenic processes or pharmacological responses to therapeutic intervention. It is considered that the telomere length is the
weak biomarker (with poor predictive accuracy), and there is currently no reliable biomarker that meets all the necessary criteria.
Keywords: senescence; aging; biomarkers; hallmarks

Received: February 25, 2019 Accepted: June 10, 2019

Introduction
In the past two decades the field of both aging dle old age, from 76 to 90 years, the period of fur-
and senescence research has undergone a signifi- ther involution of human motor functions. Finally,
cant progress. Aging can be defined as the time- after 90 years of age, a late old age is following; it is
relating irreversible proliferative deterioration of a period of decline in human physical abilities (2).
those physiological processes of the organism that Every living organism lives in a permanent strug-
support its survival and fertility (1). The result of gle with extrinsic and intrinsic agents that can
aging processes is the progressive loss of physio- damage it. Without its own repair mechanisms, life
logical integrity and impaired function of tissues of living creatures would be extremely short, since
and organs. With prolonged human lifespan, ag- the accumulation of harmful substances would
ing also moves towards the older age. Recently, el- damage the cellular elements, their function,
derly age was classified into three periods: elderly which would ultimately result in damage to the
or early old age, senile or middle old age and late various tissues and accelerated aging of the entire
old age (or long-livers). Early old age ranging from organism.
60 to 75 years is the period of initial involution of
Most of the aging definition involves a gradual,
human physical capabilities. Then follows the mid-
heterogeneous impair in the structure, function,

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Dodig S. et al. Senescence and aging

and maintenance of repair systems of various or- inhibited (5). While activation of autophagy causes
gans and an increased inclination to various dis- inhibition of apoptosis, its inhibition increases sus-
eases. One could say that the age/aging phases ceptibility to oxidative damage of the cell and ap-
are easy to recognize, but the mechanisms respon- optosis. Prolonged autophagy is associated with
sible for the aging process are difficult to define cell death. Autophagy becomes defective during
and harder to prove. Technological progress has ageing and especially in patients with age-related
established various methodological approaches diseases, since degraded molecules and orga-
to detect some cellular and molecular mecha- nelles accumulate in cells. Hence, defective au-
nisms associated with aging. Among others, scien- tophagy is a feature of old cells (7). Schematic de-
tists have focused recently on senescence (cellular piction of the aging process, with possible thera-
aging, biological aging) mechanisms as one of the peutic interventions is shown in Figure 1.
key factor in a complex aging process (3,4).
This review focuses on human senescence and ag- Senescence
ing processes, and their mechanisms. Particular at-
tention was directed to hallmarks of these pro- Senescence (from the Latin word „senex“, mean-
cesses and their possible biomarkers. In search of ing growing old) is an irreversible form of long-
scientific and review papers on the PubMed free term cell-cycle arrest, caused by excessive intracel-
search engine, the following key words were used: lular or extracellular stress or damage (12). In order
lifespan, aging, systems biology, senescence, hall- to avoid malignant transformation after the stress-
mark, markers of aging, biomarkers, biomarkers of or’s activity, cellular senescence refers to the arrest
senescence, senescence testing, and bioinformat- in the G1 phase of the cell-cycle (5). Senescent cells
ics. Epidemiological and clinical researches were are however functionally and metabolic active as
studied primarily on older people, regardless of changes occur, for example change of degrada-
their ethical affiliation. Also, animal models of ag- tion pathways of proteins, enhanced mitochondri-
ing investigation were studied. Abstracts, reports al metabolism, energy generation, etc. (13). The
from meetings and case control studies were ex- purpose of senescent cells arrest is to limit the pro-
cluded. Articles published in English between liferation of damaged cells (e.g. the spread of dam-
1997 and 2019 were included. Articles were select- age to the next cell generation), to eliminate accu-
ed according to relevance to the topic. mulated harmful factors and to disable potential
malignant transformation (5-8). In young tissues,
Three different responses that have protective role
transient senescence has beneficial effect. The
in response to cellular stressors are apoptosis (pro-
good example is the beneficial effect of senes-
grammed cell death), autophagy (from the Greek
cence to pregnancy that implies proper foetal de-
noun „autóphagos“, meaning self-devouring) and
velopment and time of parturition. A detrimental
senescence (irreversible arrest, that limits the pro-
effect refers to reproductive capacity since it caus-
liferation of damaged cells) (5-8). It seems that the
es the decrease in the number of ovarian follicles,
cellular response depends on the type of cell that
and in later age senescence causes decline in ovar-
is subjected to the harmful effect of the stressor.
ian and uterine function (14). Healthy senescence
While damaged lymphocytes tend to undergo ap-
may be accelerated by elevating the concentration
optosis, damaged epithelial cells and fibroblasts
of oxygen or various toxic substances (15). Factors
tend to undergo senescence (5). Autophagy im-
that slow down damage accumulation delay the
plies a lysosome-mediated cell’s own components
senescence.
bulk degradation and clearance (5,9). The relation-
ship between autophagy and apoptosis is com- Based on kinetics of cell senescent processes there
plex. It is not yet clear which factor determines are two main categories of senescence, i.e. acute
whether cells will die with apoptosis or with other (transient) and chronic (persistent) senescence
mechanisms. It seems that autophagy could be (16). Acute senescence is the part of normal bio-
conducive to cell death in cases when apoptosis is logical processes, and has beneficial effect within

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Dodig S. et al. Senescence and aging

Dysfunctional
Replicative telomerase
stress
ROS
Oncogenes DNA
damage
p53 p16
Prevention of senescent triggers
p21

Cellular damage

Healthy cell

Apoptosis Autophagy
? ?

Acute senescence Mixed Chronic senescence

Acute specific stress Gradually increase in stress/damage
Targeting specific cells Not targeting specific cells
Scheduled clearance Unscheduled clearance

Senescent pre-senescent
cell cell
?
Immuno- Immuno- SASP inhibition
clearance clearance
Senescent
Development cell
Injury repair Senescent cells killing
Wound healing

Late senescent
cell

Cancer Aging
Age-related diseases

Figure 1. Overview of the process of senescence and its contribution to aging of entire organism (adapted according to references
5, 10 and 11). Based on kinetics of cell senescent processes there are two main categories of senescence – acute (programmed, tran-
sient) and chronic (not programmed, persistent) senescence. While acute senescence leads to embryonic development, wound heal-
ing and tissue repair of specific populations of cells and tissues, chronic senescence that is not directed towards specific cells leads
into a stable cell-cycle arrest, a state that limits the proliferation of damaged cells. The main mediator of acute senescence is SASP. It
seems that, because of age-related immunodeficiency or less production of proinflammatory SASP factors, immune cells becomes
inefficiently in the elimination of senescent cells. p53, p16 and other tumour suppressor pathways mediators leads to senescence.
Cancer development will occur if pre-senescent cells (stressed cells) would not been removed by specific mechanisms. However,
it is not known which mechanisms are responsible for direction to senescence, apoptosis or to autophagy. Production of SASP fac-
tors may be inhibited by the use of: nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), interleukine 1α blockers,
rapamycin, metformin; senescent cell killing may be induced by natural killer cells, T cell targeting, antibodies or antibody-mediated
drug delivery. Early in life, senescent cells are transiently present and have a beneficial effect on development, homeostasis, and
regeneration. However, at a later age, senescent cells accumulate and produce detrimental effects. ROS – reactive oxygen species.
SASP – senescence-associated secretory phenotype. p53 – cellular tumour antigen p53. p21 – cyclin-dependent kinase inhibitor 1,
cell-cycle inhibitor. p16 – cyclin-dependent kinase inhibitor 2A, multiple tumour suppressor 1.

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Dodig S. et al. Senescence and aging

tissues during embryonic development, wound resistance of senescent cells to immune clearance.
healing or tissue repair. Myofibroblasts have an im- Chronic senescence has detrimental effects within
portant role during acute senescence, because cells and tissues. The knowledge that senescence
they promptly undergo senescence, limiting ex- can cause age-related diseases has instigated re-
cessive fibrosis at the site of cell/tissue damage. searchers to develop drugs that can eliminate se-
Acute senescence may be a part of programmed nescent cells. These medications could improve
mechanism of fibrosis control during tissue repair health in the elderly (Figure 1) (11,20).
(17). Acute senescent cells are eliminated through Senescent cells in elderly are not able to maintain
activation of senescence-associated secretory neither physiological tissue functions nor tissue re-
phenotype (SASP) factors and consequently acti- pair, including autophagy, whose capacity de-
vated immune clearance. Senescent cells, still met- clines with aging (7,21,22). Cellular senescence is
abolically active, found primarily in tissues with followed by senescent cell clearance within those
chronic inflammation and in renewable tissues, processes that are considered beneficial. However,
are able to create an inflammatory microenviron- if the elimination of senescent cells does not occur,
ment, to recruit phagocytic cells for elimination of senescent cells accumulate and can lead to cancer
senescent cells and finally, to promote tissue re- and aging. Investigations on animal samples have
moval. They secrete a variety of different mole- shown that senescent cells accumulate in old ani-
cules to communicate with adjacent cells. Senes- mals in leukocytes and intestinal crypt entero-
cence is enabled with the acquisition of SASP fac- cytes, in dermal fibroblasts, hepatocytes, osteo-
tors, such as interleukins (the most prominent is cytes (23).
interleukin-6, IL-6), chemokines, growth factors
(e.g. insulin-like growth factor, IGF) and regulators, Unlike apoptosis in which phagocytes remove
proteases (e.g. matrix metalloproteinases - MMPs, cells without causing inflammation, senescent cell
serine proteases), etc. (8,18,19). Released SASP fac- survive because of stimulation of the inflammato-
tors are involved in sensitizing non-senescent ry environment and removal of harmful com-
neighbouring cells to senesce, cell proliferation, pounds (24). Senescence-associated beta-galac-
disruption of normal tissue structure and function, tosidase (SA-β-GAL), is an isoform of the beta-ga-
immunomodulation (immune cells clearance), an- lactosidase enzyme, normally responsible for the
giogenesis, disabling or fostering of cancer breakdown of beta-galactosides. Its activity is pre-
growth. SASP factors have beneficial role during sent in lysosomes of senescent cells. Increased ac-
embryogenic development, accelerating wound tivity of SA-β-GAL is considered to be an outcome
healing, after tissue injury (by limiting fibrosis), in- of senescence (7).
volved in the amplification and spread of senes- Factors leading to senescence
cent cells, during suppression of tumorigenesis by
promoting the elimination of senescent cells. The Senescence can be triggered e.g. by oxidative
main function of SASP is to eliminate senescent stress, telomere damage/shortening, DNA dam-
cells. If there were no senescent cell clearance as in age, mitochondrial dysfunction, chromatin disrup-
case in elderly people, senescent cells would accu- tion, inflammation, epigenetic dysregulation, and
mulate, which would have detrimental conse- oncogene activation (17,25-27).
quences implying structural, degenerative, irrepa-
Oxidative stress
rable tissue damage and fibrosis (7,20). Chronic se-
nescence is induced through prolonged period of It is known that senescent phenotype may be
cellular stress or slow macromolecular damage stimulated/induced by various types of stresses,
(10,16). Complex effector pathways involved in including that induced by reactive oxygen species
chronic senescence significantly differ from path- (ROS). Reactive oxygen species are a natural by-
ways in acute senescence, due to large SASP het- product of the normal oxygen metabolism. It is
erogeneity involved in chronic processes and high considered that ROS regulate several physiological

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functions, like signal transduction, gene expres- function of telomeres is to protect the chromo-
sion and proliferation. The major cellular sources somes from degradation rearrangements, end-to-
of ROS are mitochondria, cell membranes and en- end fusions, and chromosome loss (36). Shorten-
doplasmic reticulum (28). While lengthening of or- ing occurs at each cellular division but is counter-
ganismal lifespan is associated with low ROS con- acted by telomerase. Telomerase is an enzyme
centration, senescent phenotype maintenance is complex that maintains telomere length. It is con-
endangered with high ROS concentrations (29). sidered that telomeres participate in the protec-
The oxidant/antioxidant imbalance causes a struc- tion of ends of chromosomes from constitutive ex-
tural damage of macromolecules (DNA, proteins posure to the DNA damage response (37). Tel-
and lipids). Age-related accumulation of damaged omere length progressively shortens with replica-
macromolecules is one of mechanisms that con- tion of nuclear DNA during mitosis, or with oxida-
tribute to the aging processes. The balance be- tive stress or with senescence and aging (38).
tween oxidant generation and antioxidant pro- While the length of the telomere at birth is about
cesses in healthy tissues is maintained with a pre- 11 to 15 kb in elderly it is significantly shorter,
dominance of various antioxidants (30,31). about 4 kb (39-42). So, senescence is mostly trig-
Reactive oxygen species of endogenous or exoge- gered when the length of the telomere shorten
nous origin induce and firm the senescent pheno- from 5–20 kb to 4–7 kb (43). The shortening of the
type by a process that involves the response to telomeres that occurs during normal aging is con-
DNA damage, epigenetic regulation and tumour trolled by the activity of specialized enzyme tel-
suppression pathway activation (e.g. cell cycle con- omerase (27). However, the balance between tel-
trol related proteins: p53 (cellular tumour antigen omere shortening and counteracting by telomer-
p53), p21 (p21Cip1, cyclin-dependent kinase inhibi- ase is disrupted during accelerated senescence as
tor 1), pRB (retinoblastoma protein). These mecha- a result of the disease.
nisms, more specifically SASP factors of senescent
cells, on the other hand, can stimulate positive DNA damage
feedback loop and result in increased ROS, espe- Critically short telomeres are recognized as DNA
cially mitochondrial ROS (mtROS) (32). As mito- damage, which trigger a DNA damage response
chondria are the main place of ROS creation, inves- (DDR). The DDR arrests cell cycle progression until
tigations have shown that mitochondrial dysfunc- damages are repaired. However, senescent cells
tion is associated with senescence, and conse- display persistent DDR foci that that are resistant
quently with the aging process. It is considered to endogenous DNA repair (44).
that mtROS and oxidative stress in general can
stimulate telomeres shortening and dysfunction, Mitochondrial DNA damage
which is one of the characteristics of aging (33). In Mitochondria are intracellular source of oxygen.
addition to ROS, as senescence inducers, other mi- Functional mitochondria regulates cellular home-
tochondrial-related effectors are also considered, ostasis through the maintenance of redox balance,
for example, redox changes, changed metabolism which implies a balance between oxygen uptake,
(34,35). ATP production, membrane potential and genera-
tion of ROS (45). Mitochondria that accumulate in
Telomere shortening senescent cells show increased concentrations of
Telomeres (from the Greek nouns „telos“ meaning ROS and increased rate of senescent cells in the
end and „merοs“ meaning part), specialized DNA- same tissues, resulting in mitochondrial dysfunc-
protein structures of human chromosomes, com- tion (27,45).
posed of several kilobases (kb) of simple repeats
(TTAGGG)n are located at the ends of chromo- Tumour suppressors and cell cycle inhibitors
somes. The length of telomeres is an accurate pre- Today, several suppressors and cell cycle inhibitors
dictor of the replicative ability of cells. The basic are known, e.g. p16 (known as cyclin-dependent

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kinase inhibitor 2A, multiple tumour suppressor 1), tions on biochemical level: „Why does human be-
p53, p21, p15 (p15INK4b, protein kinase; cyclin-de- ing (and all living organisms) age?“ and „How do
pendent protein serine/threonine kinase inhibitor, organisms age?“. Consequently, there are a large
multiple tumour suppressor), p27 (cyclin-depend- number of aging theories that are classified in a
ent kinases regulator), ADP-ribosylation factor variety of ways. For example, one of classifications
(ARF), hypophosphorylated retinoblastoma pro- theories includes the evolutionary and causality
tein (7,11). Activation of the tumour suppression theories (48). Evolutionary aging theories, that are
pathways p53 and p21 and the p16/retinoblasto- focused on the failure of natural selection to affect
ma protein pathways occurs during senescence. late-life traits, refer to programmed aging (assisted
Activation is triggered by the DNA damage, which death), non-programmed aging and senemorphic
may be result of telomeric and non-telomeric DNA aging (maladaptive aging, secondary aging). Cau-
damage or oxidative stress (27). sality theories imply the influence of the environ-
mental conditions on cellular senescence and ulti-
Characteristics of senescent cells mate death. The main role was given to telomeres
Senescent cells are characterised by flattened and shortening, free radicals damages, spontaneous
enlarged morphology. They exhibit several molec- errors, glycation end-products (48). There are also
ular markers, including telomere-dysfunction-in- theories that attempt to explain the aging process
duced foci, senescence-associated heterochroma- itself - on the one hand there are theories consid-
tin foci (SAHF), lipofuscin granules, DNA scars, al- ering the senescence as programmed processes;
tered gene expression (5,7). Another important other theories, e.g. „DNA damage theory of aging“
feature of senescent cells is release of SASP factors are focused on the accumulation of damage as the
(19). As the senescent cells are characterized by the main cause of biological aging (22,49).
irreversible growth arrest in either G1 or G2/M Aging is an intrinsic feature of all living beings. The
phase of the cell cycle, they are no longer able to complex process of biological aging is the result of
divide. These cells have special biochemical char- genetic and, to a greater extent, environmental
acteristics, e.g. the absence of proliferative Ki-67 factors and time. It occurs heterogeneously across
protein, activity of senescence-associated multiple cells and tissues. As the rate of aging is
β-galactosidase (SA-β-GAL), expression of tumour not the same in all humans, the biological age
suppressors and cell cycle inhibitors (7,11). Nuclear does not have to be in accordance with the chron-
and mitochondrial DNA damage accelerate senes- ological age. Many age-associated changes and
cence. As long as the repair mechanisms are effec- hallmarks are evident in the human body. The
tive, the cell damage can be repaired. Otherwise, changes associated with old age can be divided
when some of the repair mechanisms fail, damaged into a few categories: normal aging, somatic dis-
DNA will accumulate, obstructing cellular function eases and multiple chronic conditions, psychologi-
and causing its senescence. Inducers of senescence, cal, cognitive and social changes (50). Normal ag-
such as telomere shortening, toxic agents or onco- ing implies sensory changes (visual acuity, hearing
genes, cause the formation of SAHF, that contain loss, dizziness), muscles weakening and reduced
heterochromatin-forming proteins, such as hetero- mobility ability, fat changes. At the same time the
chromatin protein 1 (HP1) proteins, di- or tri-methyl- body increasingly succumbs to some diseases, in-
ated lysine 9 of histone H3 (H3K9Me2/3) and his- cluding hypertension, cardiovascular diseases, dia-
tone H2A variant (macroH2A) (46,47). All these cel- betes, osteoarthritis, osteoporosis, cancer, and
lular characteristics can be considered as hallmarks several neurological disorders. In elderly there are
(or possible biomarkers) of senescence. several functional changes of respiratory system
such as reduction of vital capacity, increased resid-
Aging
ual volume, reduction of pulmonary diffusion, in-
Aging has been the focus of researchers for many creased arterial-alveolar oxygen gradient, hypoxia,
years. Scientists are trying answer two basic ques- hypercapnia, increased percent of neutrophil

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granulocytes, increased ratio of CD4+/CD8+ cells in Immune system and aging

bronchoalveolar lavage fluid and decreased level The weakening of unspecific innate and highly
of antioxidant compounds (i.e. superoxide dis- specific acquired immunity takes place through
mutase, glutathione, catalase, metal binding pro- the aging of human cells (Table 1). The phagocytic
teins, vitamins C and E) (51,52). In addition, there is function is reduced, while, chemotaxis may be
a decreased number of functional glomeruli, de- conserved, especially in the presence stimulants of
creased rate of glomerular filtration and renal the complement fragment C5a (57). The number
blood flow (53). Occurrence of electrolytic distur- of macrophage precursors is decreased, the
bances (e.g. hyper- or hyponatremia) may worse phagocytic function is reduced, neutrophil dys-
other comorbidities (54). Also, there is a decrease function is observed, and naive lymphocytes pro-
in basal metabolism, the change in gastrointesti- duce less IL-2, the number of CD8+ lymphocyte in-
nal system, as well as in the hypothalamic-pitui- creases. The senile age is characterized by a high
tary-adrenal systems. The later results with low re- expression of CD25 and FOXP3 (a transcriptional
sponse to stimulation of this axis (54). In the back- factor that is crucial for the function of Treg cells),
ground of all the changes that occur during aging and increased number of CD4+/FOXP3 lympho-
are three key factors – inflammation, immune ag- cytes, changed T17/Treg ratio. All these changes
ing and senescence. are responsible for the appearance of inflammato-
Inflammation and aging ry and autoimmune diseases (60). Impaired NK
function of natural killers (NK) is associated with
Unlike acute (transient) inflammation in which the an occurrence of infective, atherosclerotic and
causative agents are removed and the damaged neurodegenerative diseases. As the thymus exhib-
tissue is cured, chronic inflammation persists for a its degenerative changes, impaired function of
long time. During chronic inflammation affected both, B cells and T cells leads to imbalance be-
tissues are infiltrated with macrophages and lym- tween inflammatory and anti-inflammatory mech-
phocytes. In addition, fibrous and necrosis of the anisms. Frequent infectious diseases in old age are
affected tissue may occur (18,55). Chronic inflam- a result of impaired function of the innate and ac-
mation is associated with many age-related physi- quired immune system. Immune system fails to
ologic or pathophysiologic processes and diseas- clear infectious antigens, infected cells, senescent
es. In normal, healthy aging, serum concentrations cells, and malignant transformed cells (56,61). Im-
of pro-inflammatory cytokines (IL-1, IL-2, IL-6, IL-8, munological changes in elderly, based on the de-
IL-12, IL-15, IL-17, IL-18, IL-22, IL-23, tumour necrosis cline of the functional capacity of the immune sys-
factor alpha – TNF-α, and interferon-gamma – tem, result in reduced resistance to infections, in-
IFN-γ) are significantly increased in comparison creased appearance of neoplasia, and increased
with younger individuals (56-58). At the same time, production of auto-antibodies responsible for the
in elderly people concentration of anti-inflamma- occurrence of autoimmune diseases (62).
tory cytokines (interleukin-1 receptor antagonist –
IL-1Ra, IL-4, IL-10, IL-37, transforming growth factor As individuals of the same age do not have the
beta 1 – TGF-β1) are higher than in young persons. same rate of age, there is a need to find specific
The role of anti-inflammatory cytokines is to neu- hallmarks that could objectively determine the
tralize pro-inflammatory cytokine activity, reduce rate of age of a person. These biomarkers might
chronic inflammation, and thus act protectively on be a valuable measure of physiological/biological
tissues. In the case of healthy aging, a balance be- age. Still, there is no universally accepted defini-
tween the action of pro-inflammatory and anti-in- tion of a biomarker of aging. Phenotypic hallmarks
flammatory mediators has been established. Their are non-invasive biomarkers, and easy to obtain
imbalance leads to aging of the body and to the (Table 2). Biochemical biomarkers can reflect some
development of various age-related pathological of the biochemical mechanisms underlying age
conditions (59). status. It would be ideal if quantitative aging bio-
markers could specifically determine the biologi-

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Table 1. Features of immune aging

Cell Features
Innate immunity
Neutrophils Reduced phagocytosis and ROS production
Reduced phagocytosis, cytokine and chemokine secretion, reduced generation of NO and
Monocytes/Macrophages
superoxide, reduced IFN-γ, inhibited response to growth factors
Reduced phagocytosis and pinocytosis, increased IL-6 and TNF-α production, diminished TLR
Dendritic Cells
expression and function
Eosinophils Reduced degranulation and superoxide production
Cytotoxic lymphocytes
NK Reduced numbers, increased reduced numbers, reduced cytotoxicity
NKT Reduced proliferation
Acquired immunity
Decreased number, reduced proliferative capacity, increased oligoclonal expansion, reduced
B cells surface MHC class II molecule expression, reduced antibody avidity, increased concentration of IgG,
IgA and concentration of autoantibodies
Reduced CD28 expression, accumulation of CD8+CD28- T cells, reduced TCR diversity, reduced signal
T cells
transduction, reduced response and proliferation, increased differentiation of CD4+ into Th17 cells
Treg Increased CD8+FOXP3+, decreased CD8+CD45RA+CCR7+
ROS - reactive oxygen species. NO - nitric oxide. NK – natural killer cells. NKT – natural killer T cell. Treg – T-regulatory cells. TCR
– T-cell receptor. IL – interleukine. IFN – interferon γ. TLR – toll-like receptor. TNF-α – tumour necrosis factor α. MHC – major
histocompatibility complex. CD – cluster of differentiation. FOXP - transcription (factor) protein. CCR – chemokine receptor.
Adapted according to references 63-66.

cal age (healthy aging) of a person, regardless of ly measured in the body or its products and evalu-
the predisposition to disease (accelerated aging) ated as an indicator of normal biological process-
(67). In laboratory medicine, organ-specific bio- es, pathogenic processes or pharmacological re-
markers imply determining those biochemical and sponses to therapeutic intervention (68,69). Thus,
haematological analytes that point to the diseases there are diagnostic, prognostic, predictive and
of particular organic systems. pharmacodynamic biomarkers.

Senescence and aging testing According to the American Federation for Aging
Research (AFAR) recommendations, aging bio-
In order to examine why and how people become markers should meet several criteria. They have to:
old with different rate, it is necessary to define the 1. predict the rate of aging (correlate with aging);
primary indicators/biomarkers of the healthy ag- 2. monitor a basic process that underlies the aging
ing process. Only in this way it will be possible to process (determine “healthy aging”, not the effects
distinguish the phenomenon of aging due to the of disease); 3. be able to be tested repeatedly with-
processes caused by various diseases that are out harming the person; 4. be applicable to hu-
commonly associated with the aging process. In mans and animals (70). However, currently, there is
this sense, the scientific community is continually no biomarker that would meet all of these criteria.
investing great efforts in discovering such bio- Scientific papers refer at biomarkers of senescence
markers. (or senescent cells) as well as at aging biomarkers.
In general, a biomarker is defined as any sub- Currently, due to the stated fact that many of the
stance, structure or process that can be objective- hallmarks do not meet biomarker definition crite-

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Table 2. Phenotypic and biochemical hallmarks of aging

Trend during
Hallmark category Hallmark subcategory Hallmark
aging
Anthropometry and
Phenotypic BMI, waist circumference I
physical function
Facial features Eye corner slope D
Nose width, Mouth width, Noise-mouth distance I
Mouth width I
Noise-mouth distance I
Biochemical Nutrient sensing (S/P) Growth hormone and IGF-1 D
Protein metabolism (S/P) Protein carbamylation, e.g. homocitruline rate I
(Erc) Glycosated hemoglobin I
(S/P) Advanced glycation end products N-glycans I
Lipid metabolism (S/P) Lipid profile, free fatty acids, isoprostanes I
Oxidative stress (Erc) superoxide dismutase D
(Erc) glutathione, glutathione reductase, glutathione
HD
peroxidase
Hormone, energy (S/P) Triiodothyronine, cortisol D
Inflammation (S/P) C-reactive protein, interleukin 6 I
Organ-specific Cardiovascular system (S/P) troponin, natriuretic peptides, endothelin I
Lung (S/P) surfactant protein D I
(arterial blood) partial pressure of oxygen D
Kidney (S/U) Glomerular filtration rate D
(S/P) creatinine, urea I
Liver (S/P) ALT, AST, GGT, albumin D
Reproductive function (S/P) LH, FSH, DHEA D
Oxygen transport (B) Htc, Hb, MCV, Rtc D
(S) erythropoietin, ferritin, hepcidin D
Blood clotting (S/P) D-dimers I
(B) platelet count D
(Plt) platelet functions I
(P) Fibrinogen I
BMI – body mass index. IGF-1 – insulin-like growth factor 1, somatomedin C. S/P – serum/plasma. Erc – erythrocytes. S/U – serum/
urine. B – blood. S – serum. P – plasma. ALT – alanine aminotransferase. AST – aspartate aminotransferase. GGT – gamma-glutamyl
transferase. LH – luteinizing hormone. FSH – follicle-stimulating hormone. DHEA – dehydroepiandrosterone. Htc – haematocrit. Hb
– haemoglobin. MCV – mean cell volume. Rtc – reticulocytes. I – increased. D – decreased. HD – increased in elderly hypertensive
patients treated for their conditions. Adapted according to reference 70.

ria, it may be better to use terms a) hallmarks of The corresponding biomarker can be identified ei-
senescent cells or hallmarks of aging or b) possible ther in pro-senescent mechanisms either in anti-
biomarkers of senescence. senescent pathways. Different methods for detec-
Research on why and how the senescence goes on tion of senescence in tissue sections or in cultured
should shed more light on this intriguing process. cells (fibroblasts) are used (Table 3). It is possible to

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Dodig S. et al. Senescence and aging

Table 3. Laboratory methods used for determination of possible senescent-cell biomarkers

Analyte Method References

morphological analysis inverted phase-contrast microscope 73
cell viability tetrazolium reduction, microplate spectrophotometer 71
SASP ELISA 12,68
SAHF immunohistochemistry 12
γH2AX histochemistry 12,68
p16, p53, and p21 histochemistry, immunohistochemistry 12
SA‐β‐GAL histochemistry, immunohistochemistry, flow cytometry 12,68,79
autophagy immunoblotting 72
cell proliferation flow cytometry 73
leukocyte absolute telomere length southern blot, PCR, FISH 68,75,76
ELISA – enzyme linked immunosorbent assay. SASP – senescence-associated secretory phenotype. SAHF – senescence-associated
heterochromatin foci. γH2AX – a type of histone protein from the H2A family, a marker for activation of DNA damage response. PCR
– polymerase chain reaction. p16 – cyclin-dependent kinase inhibitor 2A, multiple tumor suppressor 1. p53 – tumour suppressor
gene, induces senescence growth arrest via activated p21–p53 pathway. p21– cell-cycle inhibitor, induces senescence growth arrest
via activated p21–p53 pathway. SA‐β‐GAL – senescence-associated β-galactosidase. FISH – fluorescent in situ hybridization.

conduct morphological analysis of senescent cells, marker for activation of DNA damage response),
detection of intracellular SAHFs, determination of p16, p53 (induces senescence growth arrest via ac-
cell viability, p21 detection and measuring SA‐β‐ tivated p21–p53 pathway), and p21 concentrations
GAL activity, the ability of autophagy, cell prolifer- (induces senescence growth arrest via activated
ation, leukocyte absolute telomere length by p21–p53 pathway) (12). Telomere attrition is the in-
southern blot analyses of the terminal restriction trinsic property of healthy cellular aging, and is
fragments, by quantitative polymerase chain reac- also associated with many age-related diseases,
tion or quantitative fluorescence in situ hybridiza- like atherosclerosis, myocardial infarction, heart
tion (71-76). failure, Alzheimer’s dementia (76). For more than a
A lysosomal hydrolase, SA-β-GAL, normally active decade telomere length has (most often average
at pH 4, in senescent cells is active at pH 6. How- leukocyte telomere length) been postulated as a
ever, the SA-β-GAL, is present not only in senes- biomarker of human aging (80).
cent cells but also in presenescent, quiescent or These possible biomarkers are detected separately
immortal cells (77). It may be detected in tissue in consecutive sections; it means that multiple
sections histochemically and immunohistochemi- possible biomarkers are not determined within
cally (12,78). Conventional SA‐β‐GAL staining fails the same cells. Although it was confirmed in
to distinguish between different cell types that mouse tissues that most possible markers increase
can be a source of senescent cells within complex with age, there is still insufficient data that would
tissues, limiting our understanding of the underly- refer to healthy human tissues (77). Telomere
ing biological phenomena. (73,79). As the single length measurement is emerging as a tool that
parameter is not enough to define with confi- may have implications for prevention, disease
dence that cells are senescent, SA-β-GAL staining monitoring, and intervention development. It has
may be combined with additional possible bio- been a subject of debate whether telomere length
markers, e.g. SASP factors, SAHF formation, γH2AX is a biomarker of aging in specific tissues or for a
(a type of histone protein from the H2A family, a whole organism, since the aging of different tis-

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Dodig S. et al. Senescence and aging

sues and organs of the human body is not the the hallmarks and biomarkers of senescence and
same (3,81). Therefore, In human aging, telomere aging. The knowledge of the mechanisms of se-
length is a weak biomarker with poor predictive nescence and the influence of senescence on ag-
accuracy. Glycans might be a better possible bio- ing of organism have evolved due to the develop-
marker of chronological and biological age than ment of numerous standard and sophisticated
telomere lengths (81,82). Histochemical staining of and laboratory methods. Senescence and aging
lipofuscin (i.e. lipid - containing lysosomal gran- can be observed from different aspects so that
ules) of paraffin sections has been shown to be this topic can be observed in the context of re-
one of the possible markers of senescence in age- search of mainly human fibroblasts, leukocytes,
related diseases (83,84). Recently a new method cell cultures and animal leukocytes and intestinal
for the determination of lipofuscin in liquid sam- crypt enterocytes, dermal fibroblasts, hepato-
ples of stressed or damaged cells was introduced cytes, osteocytes, computational biology meth-
(85). Mass cytometry method, as a method that ods, the examination of factors involved in the
combines flow cytometry and mass spectrometry, normal pathways of acute and chronic senescence,
enables the simultaneous quantification of numer- diseases that can affect the process of senescence,
ous cellular parameters (SA-β-GAL) at single-cell processes that can repair senescence effects
resolution (86). Also, among potential predictors (5,7,10,11,16,17,21-23,27,43,50,78,81,88,89), etc. In or-
of biological age could be included the degree of der to successfully investigate these processes, it is
methylation of DNA, transcriptomic predictors, necessary to find standardized biomarkers of se-
proteomic predictors, metabolomics-based pre- nescence or the healthy aging of the organism
dictors, and composite biomarker predictors (87). (70). It is important to know the extent of deter-
Additional research is needed to confirm that gly- mining a particular biomarker to prevent age-re-
cans or some other compounds will meet neces- lated assessment of the entire organism. Standard-
sary criteria to be the biomarkers of senescence. In ized biomarkers could also help in the monitoring
the future, biomarker and therapeutic target can- of therapeutic interventions in the process of se-
didates will be examined for a follow-up study, nescence, which is one of the goals of examining
which will facilitate longitudinal monitoring of all aspects of senescence (11,21).
therapeutic interventions on senescence and ag-
ing. Instead of a conclusion
Today, the bioinformatics, as an interdisciplinary
• The largest number of study of senescence and
field of science, helps to analyse and interpret bio-
aging processes were made on cell cultures
logical data on aging and senescence, including
and animal models.
studies of gene expression and comparative and
• The senescence seems to be a critical factor in
pathway analyses (88-90). Computational biology
both the normal aging process and pathologies
of aging refers to a wide range of data, from de-
associated with aging.
mographic to genomic transcriptomic, proteomic
and metabolomic studies (88). CSGene database • There are currently no standardized biomarkers
has been developed for exploring cell senescence („gold standard“) of cellular aging process or
genes and to highlight the roles of cell senescence the healthy aging of the organism. Biomarkers
genes in the control of rRNA gene transcription described in literature do not meet all criteria of
(89). an ideal aging biomarker and actually repre-
sent various hallmarks of the aging process.
Between 1997 and 2019, PubMed published about
• Most biomarkers currently being examined as
363,000 articles on senescence and aging, and in
senescence or aging biomarkers are related to
the first four months of 2019, more than 10,000 ar-
age-related illnesses rather than the process of
ticles. In this review, 90 articles have been selected
healthy aging.
to help us better understand the need to discover

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Dodig S. et al. Senescence and aging

• As the effector mechanisms of senescence are ers) and the possible use of appropriate thera-
neither necessarily specific to senescence nor py intervention (pharmacodynamic biomark-
present in all forms of senescence (the rate of ers).
senescence is not the same for all types of cells), • The identification and selection of reliable
the interpretation of existing biomarkers of se- biomarker(s), and the use of reproducible meth-
nescence (for now the hallmarks or possible bi- ods could help to better understanding of com-
omarkers) should be context dependent. Addi- plex web of senescence and aging processes,
tionally, a combination of multiple biomarkers but it will also open some new questions.
should be used. • Despite new findings at the cellular and molec-
• Detection of biomarkers, in particular their ular level the understanding the aging process
quantification and validation, are necessary for is still limited.
understanding the senescence processes (diag-
nostic biomarkers), monitoring of the rate of se- Potential conflict of interest
nescence (prognostic and predictive biomark- None declared.

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