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Age-related degeneration of articular cartilage in the pathogenesis of osteoarthritis: Molecular markers of senescent chondrocytes

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Background: Our previous studies demonstrated the expression of procollagen11A1 in fibroblasts of pancreatic cancer desmoplasia and the lack of expression in fibroblasts of pancreatitis by means of the polyclonal antibody (anti-proCOL11A1 pAb) we generated. In a similar way, we decided to compare the expression of procollagen11A1 in fibroblasts of infiltrating ductal carcinoma of the breast and fibroblasts of benign sclerosing lesions of the breast, in order to validate the anti-proCOL11A1 pAb in this setting and to study how proCOL11A1 expression relates to other prognostic and predictive factors, as well as to survival. Methods: 45 core biopsies of sclerosing adenosis and 50 core biopsies of infiltrating ductal carcinoma of the breast were stained with anti-proCOL11A1 pAb, a polyclonal antibody highly specific to the less homologous fraction of proCOL11A1 (in comparison with proCOL5A1 and proCOL11A2). In addition, the expression of the proCOL11A1 gene was measured by RT-qPCR. On the other hand, the expression of proCOL11A1 was compared to the expression of estrogenic receptors, progestagen receptors, the state of the epidermal growth factor receptor 2 (HER2), the histologic grade and the stage of the disease. We also compared the immunohistochemical expression of proCol11A1 to the disease-free interval, and to overall survival. Results: The immunohistochemical analysis showed that proCOL11A1 was expressed in 100% of infiltrating ductal carcinomas, but only focally expressed in 2,2% (1 case) of sclerosing adenosis, in agreement with RT-qPCR results. ProCOL11A1 expression did not prove to have a prognostic value in relation to the disease-free interval or to overall survival in infiltrating ductal carcinoma. Conclusion: The anti-proCOL11A1 pAb is a stromal marker for breast cancer and the expression of proCOL11A1 does not seem to have a prognostic value in infiltrating ductal carcinoma of the breast.
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Summary. Aging is a natural process by which every
single living organism approaches its twilight of
existence in a natural way. However, aging is also linked
to the pathogenesis of a number of complex diseases.
This is the case for osteoarthritis (OA), where age is
considered to be a major risk factor of this important and
increasingly common joint disorder. Half of the world's
population, aged 65 and older, suffers from OA.
Although the relationship between the development of
OA and aging has not yet been completely understood, it
is thought that age-related changes correlate with other
risk factors. The most prominent hypothesis linking
aging and OA is that chondrocytes undergo premature
aging due to several factors, such as excessive
mechanical load or oxidative stress, which induce the so
called stress-induced senescent state, which is
ultimately responsible for the onset of OA. This review
focuses on molecular markers and mechanisms
implicated in chondrocyte aging and the pathogenesis of
OA. We discuss the most important age-related
morphological and biological changes that affect
articular cartilage and chondrocytes. We also identify the
main senescence markers that may be used to recognize
molecular alterations in the extracellular matrix of
cartilage as related to senescence. Since the aging
process is strongly associated with the onset of
osteoarthritis, we believe that strategies aimed at
preventing chondrocyte senescence, as well as the
identification of new increasingly sensitive senescent
markers, could have a positive impact on the
development of new therapies for this severe disease.
Key words: Osteoarthritis, Aging, Cartilage,
Chondrocyte, Lubricin, Senescence, Senescence markers
Introduction
Our body is made up of an incredibly large number
of cells, around 100 billion, some of them have a rather
short life, others such as chondrocytes remain for a
lifetime, but at a certain point, the mechanism slows
down, cell duplication starts to fail and the cells are no
longer replaced by other ones. The cells arrest their cell
cycle progression. Each cell has a limited number of
possible divisions, which is fixed between 50 and 70.
The reason lies in the structure of chromosomes that are
duplicated at each division in order to obtain one copy
for itself and another for the daughter cell that will play
the same role in the body. The division process,
however, is not perfect, the cellular mechanism fails to
copy the ends of chromosomes, the so-called telomeres
(Fig. 1). After each cell division, the telomeres become
shorter and shorter and are eventually completely worn
out and parts of chromosomes that contain essential
genetic information begin to erode. At this point the cell
is at the end of its life and it approaches death by
Review
Age-related degeneration of articular
cartilage in the pathogenesis of osteoarthritis:
molecular markers of senescent chondrocytes
Giuseppe Musumeci
1
, Marta Anna Szychlinska
1
and Ali Mobasheri
2,3,4
1
Department of Bio-medical Sciences, Human Anatomy and Histology Section, School of Medicine, University of Catania, Catania,
Italy,
2
School of Veterinary Medicine, Faculty of Health and Medical Sciences, University of Surrey, Duke of Kent Building, Guildford,
Surrey, United Kingdom,
3
Medical Research Council and Arthritis Research UK Centre for Musculoskeletal Ageing Research, Arthritis
Research UK Centre for Sport, Exercise and Osteoarthritis, Arthritis Research UK Pain Centre, University of Nottingham, Queen's
Medical Centre, Nottingham, United Kingdom and
4
Center of Excellence in Genomic Medicine Research (CEGMR), King Fahd
Medical Research Center (KFMRC), King AbdulAziz University, Jeddah,Kingdom of Saudi Arabia
Histol Histopathol (2015) 30: 1-12
http://www.hh.um.es
Histology and
Histopathology
Cellular and Molecular Biology
Offprint requests to: Dr. G. Musumeci Ph.D., Department of Bio-medical
Sciences, Human Anatomy and Histology Section, School of Medicine,
University of Catania, Via S. Sofia 87, 95125 Catania, Italy. e-mail:
g.musumeci@unict.it
a
ctivating specific mechanisms. In order to avoid the
aging process, a magic molecule able to stretch the
ends of chromosomes would be necessary. This normally
happens in cells and it is due to telomerase. However,
telomerase is active only in embryonic cells and in a few
adult cells such as cells of the immune system, in the
other ones its activity runs out over time. It is for this
reason that we talk about cell senescence. Cellular
senescence is strongly associated with the development
of several serious diseases such as cancer, diabetes,
cardiovascular and neurodegenerative diseases, and
osteoarthritis (OA) (Burnet and Berger, 2014). OA is a
degenerative joint disease, which affects especially the
articular cartilage, leading to pain and stiffness of the
affected joint. OA is one of the most disabling
musculoskeletal disorders in the world and it affects
mostly the elderly population (Musumeci et al., 2014a).
I
n this review we discuss how cellular senescence can
influence the onset of a complex joint disease such as
OA. We will discuss the most important features of
cellular senescence and how the age-related changes,
which arise at cellular and tissue level, influence the
development and progression of OA. Lastly, we will list
some of the most important senescence markers used to
evidence the senescence of chondrocytes.
Osteoarthritis
OA is the most common form of joint disease and
affects mainly hips, knees, hands, and feet, leading to
severe disability and loss of quality of life, particularly
in the elderly population (Musumeci et al., 2013a).
Indeed, half of the world's population, aged 65 and older,
suffers from OA. It has been estimated that 9.6% of men
2
Age-related degeneration of articular cartilage
Fig. 1. Telomere shortening.
A telomere is a region of
repetitive DNA (TTAGGG
repeats) at the end of a
chromosome, which protects
the end of the chromosome
from deterioration. Each time
cells divide, telomeres
shorten, and there is a limit to
the number of times a given
cell can go on dividing, the so-
called Hayflick limit. When
the length of the telomeres is
too short cell division stops.
a
nd 18% of women in that age group, have symptomatic
OA. Moreover, OA is considered the most important
cause of impaired mobility and contributes to 50% of all
the musculoskeletal diseases worldwide (Wolf and
Pfleger, 2003). The disease is characterized by joint
dysfunction due to gradual changes in several structures
of the joint such as synovium, subchondral bone and
especially articular cartilage (Fig. 2) (Buckwalter and
Mankin, 1998a). Progressive wear and tear on articular
cartilage can lead to a progressive cartilage tissue loss,
further exposing the bony ends, leaving them without
protection (Buckwalter and Mankin, 1998b). This finally
deteriorates into the most common form of arthritis, or
rather moderate OA at early stage (Fig. 3) and severe
OA at advanced stage (Fig. 4) (Musumeci et al., 2014a).
The degradation, consequent loss of articular
cartilage and formation of osteophytes lead to chronic
pain and functional restrictions in the affected joint.
Unfortunately, articular cartilage has a limited
regenerative capacity (Iwamoto et al., 2013; Musumeci
et al., 2013b). Consequently, once injured, cartilage is
much more difficult to self-heal and the only way to
improve the patient’s condition is therapeutic
intervention. Different factors can be involved in the
development of OA, such as joint injury, genetic
predisposition, defective position of joints, obesity,
malnutrition and excessive mechanical load, which all
lead to similar alterations of the articular cartilage
(
Goldring and Goldring, 2007; Lee et al., 2013;
Musumeci et al., 2014b,c). However, the prevalence of
OA rises directly with age, which represents the most
prominent risk factor for the initiation and progression of
primary OA, but it is important to underline that OA is
not a simple “wearing out in time” of the joints and the
degenerative changes related to age can be distinguished
from those due to the disease (Musumeci et al., 2013c).
The relationship between the development of OA and
aging is not completely understood and it is thought that
the age-related changes are correlated to other risk
factors, which may occur concurrently or in conjunction
with it. In reality, not all older adults develop OA and
OA-like changes can also develop without a significant
contribution of aging. Thus, aging and OA are inter-
related but not inter-dependent (Loeser, 2004). However,
there is also a possibility that the chondrocytes undergo
premature aging due to several factors, such as excessive
mechanical load or oxidative stress. In the latter case,
aging and the development of OA are both inter-related
and inter-dependent (Martin et al., 2004a; Akagi, 2010).
Cellular aging
Cellular aging, or cell senescence, refers to the
limited capacity of mitotic cells to further multiply in
time (over 30-40 divisions). This limit is known as the
Hayflick limit (Hayflick, 1984). This form of
3
Age-related degeneration of articular cartilage
Fig. 2. Healthy articular knee
cartilage from rat. Hematoxylin
and Eosin staining. Normal
articular knee hyaline cartilage
layers: superficial zone,
intermediate (or middle zone),
radial zone (or deep zone) and
calcified cartilage (or calcified
zone). Tidemark, the border
between non-calcified and
calcified cartilage. In the
superficial zone, cells are flat
and small; in the middle and
deep zone, cells are organized
in columns; the tidemark is
evident. Scale bars: 100 µm.
s
enescence is called replicative senescence, also
known as intrinsic senescence, which results from an
arrest in cell-cycle progression. Some of the changes
exhibited by cells, which have undergone replicative
senescence can be found in cells in older adults, such as
shortened telomeres, formation of senescence-associated
(SA) heterochromatin (Muller, 2009) and changes of
phenotype with an alteration in gene expression (Bodnar
et al., 1998). It has been hypothesized that the telomere
length could be considered as a marker for replicative
senescence. Telomeres cannot be completely replicated
in primary cells and become shorter with each round of
cell division. Telomeres are nucleoprotein structures
(TTAGGG repeats) that cap the ends of the linear
eukaryotic chromosomes and thereby protect their
stability and integrity during replication by protecting
chromosome ends against exonucleases (Fig. 1).
Telomeres are replicated by a special reverse
transcriptase called telomerase, in a complex mechanism
that is coordinated with the genome's replication.
Telomerase is an RNA-dependent DNA polymerase that
synthesizes telomeric DNA sequences and comprises
two essential components. One is the functional RNA
component (in humans called hTERC), which serves as
a template for telomeric DNA synthesis. The other is a
catalytic protein (hTERT) with reverse transcriptase
activity and the primary determinant for the enzyme
a
ctivity (Bryan and Cech, 1999; Kupiec, 2014). The
level of telomerase in normal human somatic tissues is
insufficient to prevent telomere shortening. Telomeres
can be lengthened through increasing telomerase activity
by exogenous expression of hTERT or hTR (the RNA
template) (Greider, 1998). As proof of this concept the
chondrocytes transduced with hTERT proved to be able
to increase telomere length and therefore to prolong cell
lifespan, increasing in this way the efficacy of cartilage
repair (Martin and Buckwalter, 2003). Another type of
senescence is “stress-induced senescence”, also known
as extrinsic senescence, which is independent of
telomere length. In quiescent cells such as chondrocytes,
this type of senescence may be more important than the
replicative version, because progressive telomere
shortening cannot completely explain senescence in
these post-mitotic cells (Ben-Porath and Weinberg,
2005; Chen and Goligorsky, 2006). The various types of
stress responsible for this kind of senescence include
DNA damage, oxidative stress, oncogene activity,
ultraviolet radiation and chronic inflammation (Itahana
et al., 2004; Campisi, 2005). Oxidative stress is thought
to play a major role as a stressor. It results when the
amount of reactive oxygen species (ROS) exceeds the
anti-oxidant capacity of the cell. ROS are generated by
intracellular enzymes such as nicotine amide adenine
dinucleotide phosphate (NADPH) oxidase and 5-
4
Age-related degeneration of articular cartilage
Fig. 3. Moderate OA articular
knee cartilage from rat.
Hematoxylin and Eosin
staining. Articular knee hyaline
cartilage layers at early OA
stage: superficial zone,
intermediate (or middle zone),
radial zone (or deep zone) and
calcified cartilage (or calcified
zone). Tidemark, the border
between non-calcified and
calcified cartilage. Clear deep
fissures in the articular surface,
the cells from the superficial,
intermediate and deep zone,
where chondrocytes are not
arranged in columns. The
tidemark is not intact in all its
extension and the subchondral
bone shows little fibrillation.
Scale bars: 100 µm.
l
ipoxygenase in response to activation of specific cell
signaling pathways (Kamata and Hirata, 1999; Finkel
and Holbrook, 2000). A direct role for increased ROS
levels in promoting cell senescence is a positive
feedback activation of the ROS-protein kinase C delta
(PK) signaling pathway, which cooperates with the
p16
INK4A
-retinoblastoma protein (Rb) pathway, which
plays an important role in the control of cell-cycle
progression (Takahashi et al., 2006). Telomere
shortening is also observed in stress-induced senescence
and it is due to oxidative damage to DNA caused by
ROS. The ends of chromosomes are particularly
sensitive to oxidative damage, which causes telomere
erosion similar to that seen with replicative senescence
(Yudoh et al., 2005). Also, the ROS generated from
excessive mechanical loading and stimulation of
cytokines contribute to DNA damage, which
subsequently results in telomere shortening (Tomiyama
et al., 2007; Davies et al., 2008). Cellular senescence, as
well as apoptosis, can be viewed as a powerful tumor-
suppressor mechanism that withdraws cells with
irreparable DNA damage from the cell cycle (de Lange
and Jacks, 1999; Artandi and DePinho, 2000; Puzzo et
al., 2014) through the intrinsic or mitochondrial (Loreto
et al., 2011a; Caltabiano et al., 2013) and extrinsic
apoptosis pathway (Loreto et al., 2011b; Cardile et al.,
2013). Several recent studies report that cartilage
degeneration also coincides with increased apoptotic
c
hondrocytes (Musumeci et al., 2011a,b; Galanti et al.,
2013). Therefore, the senescence signals, that is, a
telomere-based one or a stress-based one, trigger a DNA
damage response and this response shares a common
signaling pathway that converges on either or both of the
well-established two tumor- suppressor proteins, p53
(the p53-p21-pRb pathway) (Martin and Buckwalter,
2003; Herbig and Sedivy, 2006) and RB and pRb
proteins (the p16-pRb pathway) (Musumeci et al., 2010,
2011c). In the p53-p21-pRb pathway, senescence stimuli
activate the p53, which then can induce senescence by
activating pRb through p21, which is a transcriptional
target of p53. This senescence can be reversed upon
subsequent inactivation of p53. In the p16-pRb pathway,
senescence stimuli induce p16, which activates pRb.
Once the pRb pathway is engaged by p16, the
senescence cannot be reversed by subsequent
inactivation of p53, silencing of p16 or inactivation of
pRb (Beauséjour et al., 2003). The difference between
these two pathways is that the p53-p21-pRb pathway
mediates the senescence due to telomere shortening and
the p16-pRB pathway is thought to mediate premature
senescence (Beauséjour et al., 2003). Once cells have
entered senescence, they are arrested in the G1 phase of
the cell cycle and display a characteristic morphology
(vacuolated, flattened cells) and altered gene expression
(Cristofalo et al., 2004). The senescent cells exhibit the
so-called “senescent secretory phenotype” (SSP), which
5
Age-related degeneration of articular cartilage
Fig. 4. Severe OA articular
knee cartilage from rat.
Hematoxylin and Eosin
staining. Articular knee
hyaline cartilage layers at
advanced OA stage, due to
aging: superficial zone,
intermediate (or middle zone),
radial zone (or deep zone)
and calcified cartilage (or
calcified zone). Tidemark, the
border between non-calcified
and calcified cartilage. Cells
are arranged in clusters
especially around fissures or
disappear completely as the
disease progresses. The
organization of cartilage is
completely disordered and
replaced by fibrocartilaginous,
scar-like tissue with fibroblast
like cells. Scale bars: 100 µm.
c
ould be also correlated with the development of OA. It
is interesting to note that the senescent cells, which are
mitotically inactive, are biologically active (Campisi,
2005). These cells are able to increase the expression of
genes that inhibit proliferation and to increase the
secretion of several proteins, including inflammatory
cytokines such as interleukin-6 (IL-6) and interleukin-1β
(IL-1β), degradative enzymes such as metalloproteinases
(MMPs) and growth factors such as epidermal growth
factor (EGF) that regulate cell proliferation and all of
which may stimulate tissue aging and tumorigenesis
(Zhang et al., 2003). Recent studies reported that the
increased expression of the IL-8 receptor CXCR2 and
insulin-like growth factor binding protein 7 (IGFBP7)
could also contribute to cell aging (Acosta et al., 2008;
Wajapeyee et al., 2008). The accumulation of cells
expressing the SSP can also contribute to tissue
senescence by impairing the extracellular matrix due to
the increased secretion of degradative enzymes (Campisi
and dAdda di Fagagna, 2007). Another important
feature of senescent cells is represented by the epigenetic
changes related to the formation of foci of
heterochromatin, referred to as senescence-associated
heterochromatin foci (SAHFs), which include histone
variants such as the macro-H2A (Zhang and Adams,
2007).
Age-related articular cartilage degeneration
Articular cartilage matrix undergoes several changes
with age, including structural, molecular and mechanical
ones, surface fibrillation, alterations in structure and
composition of proteoglycans, increased collagen cross-
linking and decreased tensile strength and stiffness
(Hollander et al., 1995; Squires et al., 2003) (Fig. 5).
Deterioration in chondrocyte function accompanies these
changes also in the extracellular matrix (Aurich et al.,
2002). Several reports revealed that chondrocyte
senescence contributes to the risk for cartilage
degeneration by the decreased ability of chondrocytes to
maintain and repair the articular cartilage tissue (Martin
and Buckwalter, 2001a; Aigner et al., 2002). There is
clinical evidence from Magnetic Resonance Imaging
(MRI) studies that the articular cartilage in the knee
thins with aging, especially at the patella and at the
femoral side of the joint (Hudelmaier et al., 2001; Ding
et al., 2005). The progressive articular cartilage thinning
with age is related to gradual loss of cartilage matrix and
decrease in cartilage hydration and cellularity. This kind
of damage stimulates a chondrocyte specific synthetic
and proliferative response that may maintain or even
restore the articular cartilage. This response may
continue for years. However, in instances of progressive
joint degeneration the anabolic response eventually
declines and the imbalance between chondrocyte
synthetic activity and degradative activity leads to
progressive thinning of articular cartilage. These
alterations may further accelerate the loss of articular
cartilage (Buckwalter et al., 2000). Different changes
o
bserved in articular cartilage with aging are probably
due to chondrocyte senescence, which results in the
progressive decrease in cell function. In fact, the mitotic
and synthetic activity of human chondrocytes decline
with age. They become less responsive to anabolic
mechanical stimuli, to anabolic cytokine and to insulin-
like growth factor I (IGF-I). The cells synthesize smaller
aggrecans and less functional link proteins leading to the
formation of smaller and more irregular proteoglycan
aggregates. The latter is the most striking change in
articular cartilage matrix related with age. Aggrecans are
the molecules that give articular cartilage its stiffness to
compression, resilience and durability, thus their
alteration makes the tissue more vulnerable to injury and
development of progressive degeneration (Buckwalter et
al., 1986; Buckwalter and Rosenberg, 1988; Bolton et
al., 1999; Martin and Buckwalter, 2000). Moreover, the
senescent cartilage matrix appears more susceptible to
the accumulation of advanced glycation end-products
(AGEs) in cartilage collagen, which results in increased
cross-linking and in subsequent increased stiffness,
making the cartilage more susceptible to fatigue failure
(Verzijl et al., 2002). In addition, the increased levels of
AGEs in articular cartilage may also affect chondrocyte
function by decreasing its anabolic activity. The
mechanism proposed to be responsible for this alteration
is the expression of the receptor for the advanced
glycation end-products (RAGE) by chondrocytes, which
proves to be increased both with aging and in
development of OA (DeGroot et al., 1999; Loeser et al.,
2005). Stimulation of chondrocyte RAGE results in
increased production of MMPs and in modulation of the
chondrocyte phenotype to hypertrophy, which represent
two hallmarks of OA (Cecil et al., 2005; Yammani et al.,
2006). Furthermore, RAGE signaling is also associated
with increased levels of ROS, providing another link
between oxidative stress, aging and OA (Loeser, 2004).
Another important feature of the aged articular cartilage
is its increased calcification, as demonstrated
radiographically (Felson et al., 1989). This could be
associated with the increased activity of
transglutaminase, involved in the biomineralization
process (Rosenthal et al., 1997) and with increased
production of the inorganic pyrophosphate in response to
transforming growth factor β (TGF-β) stimulation
(Felson et al., 1989). Chondrocalcinosis is strongly
associated with OA, but there is evidence of older people
with asymptomatic chondrocalcinosis, thus it proves not
to be inter-dependent with the development of OA and
its role is not completely clear (Doherty and Dieppe,
1988; Rosen et al., 1997).
Chondrocyte senescence
Chondrocytes from older adults exhibit many
changes, typical of cell senescence, when compared with
cells isolated from young adults. The most evident
change is represented by telomere shortening,
characteristic of replicative senescence. This evidence is
6
Age-related degeneration of articular cartilage
c
ontroversial as adult articular chondrocytes rarely, if
ever, divide in normal tissue in vivo. The lack of cell
division in normal adult articular cartilage suggests that
the chondrocytes present in the cartilage of an older
adult are likely to be the same cells that were present
decades earlier. This fact makes these cells more
susceptible to the accumulation of changes from both
aging and extrinsic stress. In fact, it is most likely that
chondrocyte senescence is the extrinsic type, induced by
different stressors. The telomere shortening in adult
chondrocytes could be due to DNA damage from ROS
as discussed further above (Mankin, 1963; Martin and
Buckwalter, 2001b; Martin et al., 2004b). The increased
ROS levels could be both age-related (Del Carlo and
Loeser, 2003) and generated from excessive mechanical
loading and/or stimulation by cytokines (Kurz et al.,
2005; Davies et al., 2008). There is also evidence for
reduced levels of antioxidant enzymes in cartilage with
aging and in OA that would contribute to chondrocyte
senescence and oxidative damage. In human articular
chondrocytes, decreased levels of mitochondrial
superoxide dismutase were found both with aging and in
OA cells (Finkel and Holbrook, 2000). Moreover, it has
been hypothesized that joint injury accelerates
chondrocyte senescence and that this acceleration plays
a role in the joint degeneration responsible for post-
t
raumatic OA. Indeed, excessive loading of articular
surfaces due to acute joint trauma or post-traumatic joint
instability, incongruity or mal-alignment increases
release of ROS, and the increased oxidative stress on
chondrocytes accelerates chondrocyte senescence (James
et al., 2004). Other important features of chondrocyte
senescence are the exhibition of SSP, which has
important implications in development and progression
of OA and the decline in the proliferative and anabolic
response to growth factors, as well as their reduction in
cartilage. It has been noted that senescent chondrocytes
lose the ability in response to: IGF-I, which is known to
be an important autocrine survival factor that stimulates
cartilage matrix synthesis (Martin et al., 1997); TGF-β,
an important cartilage anabolic factor (Scharstuhl et al.,
2002) and to bone morphogenetic protein 6 (BMP-6),
known to stimulate proteoglycan synthesis (Bobacz et
al., 2003). Chondrocyte senescence also contributes to
the decline in the cell number within the cartilaginous
tissue, due to increased cell death. Several studies
demonstrated the loss of cellular density in cartilage with
aging or/and in OA (Vignon et al., 1976; Adams and
Horton, 1998; Horton et al., 1998; Aigner et al., 2004a;
Kuhn et al., 2004). These findings provide evidence to
support the concept that chondrocyte senescence may be
involved in the progression of cartilage degeneration,
7
Age-related degeneration of articular cartilage
Fig. 5. Stress-induced
senescence and
Osteoarthritis. The
telomere shortening
process in senescent
chondrocytes is more
probably due to the
stress-induced type of
senescence. Oxidative
stress and excessive
mechanical loading are
thought to be the major
stressors that induce
the increased
production of ROS,
which are responsible
for DNA damage and
for the subsequent
senescence of the
cells. Once cells have
entered senescence,
they are arrested in the
G1 phase of the cell
cycle and they display
a characteristic gene
expression called
“senescent secretory
phenotype”, which is
strongly correlated with
the development of
OA.
b
ecause of their decreased ability to maintain or restore
the articular cartilage (Fig. 5).
Chondrocyte senescence markers
An altered gene expression pattern on the cellular
level appears to be one potentially important facet of
chondrocyte behavior in OA cartilage (Aigner et al.,
2004b, 2007). The diversification of gene expression in
senescent chondrocytes is due to stochastic DNA
damage, which represents a core mechanism in cellular
aging in general and in OA cartilage degeneration in
particular. The evidence of senescence in chondrocytes
can be investigated using several senescence markers,
such as senescent-associated-β-galactosidase (SA-βgal),
highly condensed domains of facultative
heterochromatin SAHF, increased p53, p21, pRb and
p16
INK4a
(Fig. 5). Staining for SA-βgal has been shown
to be present in articular chondrocytes from older adults
and in OA chondrocytes (Martin and Buckwalter, 2001b;
Price et al., 2002). SA-βgal is related to the detection of
increased levels of the lysosomal enzyme β-
galactosidase at pH 6.0 rather than at the normal pH 4.5.
Detection of its activity at pH 6.0 is thought to be due to
an increase in lysosomal mass (Itahana et al., 2007).
Chondrocyte SA-βgal staining, as well as telomere
shortening, has also been noted after treatment in vitro
with IL-1β or H
2
O
2
consistent with stress-induced
senescence (Dai et al., 2006). Although SA-βgal is a
useful senescence marker, its activity is critically
dependent on the detection conditions, and SA-βgal is
also expressed in the non-senescent cells that have a
high lysosomal content (Kurz et al., 2000; Matthews et
al., 2006). Multiple markers of senescence are therefore
recommended to demonstrate senescence in vivo. SAHF
are thought to repress expression of proliferation-
promoting genes, thereby contributing to senescence-
associated proliferation arrest. Inclusion of proliferation-
promoting genes, such as cyclin A, into these compact
chromatin foci is thought to silence expression of those
genes, which are associated with cell cycle arrest
(Adams, 2007). Ink4a encodes an archetypical cyclin-
dependent inhibitor (CKI) associated with senescence.
I
ndeed, the over-expression of p16
INK4a
i
n chondrocytes
is associated with SSP, which includes increased
production of pro-inflammatory cytokines (such as IL-6,
IL-8, IL-1β) and matrix remodeling regulatory
metalloproteinases (such as MMP1, MMP13, etc)
(Leonardi et al., 2008; Loreto et al., 2013). As
mentioned above, all these factors are deleterious for
cartilage integrity. According to this finding, the
repressed levels of miR-24, a negative regulator of
p16
INK4a
, was also found in OA cartilage (Philipot et al.,
2014). Recently, the expression of Caveolin1, a protein
that participates in premature cellular senescence, was
also investigated in human OA cartilage. It was observed
that the treatments with catabolic factors of oxidative
stress (H
2
O
2
) and IL-1β, which simulate the OA
environment, was able to up-regulate the expression of
caveolin1. The over-expression of caveolin1 is
associated with cartilage degeneration and the mediation
of the premature senescence in OA chondrocytes by
activating p38 MA0PK, which impair the ability of
chondrocytes to produce type II collagen and aggrecan
(Dai et al., 2006). Other important senescence markers
are represented by telomere length and telomerase
activity. As discussed in detail above, telomere
shortening is the most representative feature of cellular
aging, and it is due to the decreased expression of
telomerase with time which leads to telomere instability.
Telomere length can be measured by using the Single
Telomere Length Assay (STELA), Southern blot
analysis, Q-PCR and the more recent Quantitative-
Peptide Nucleic Acid-Fluorescent in situ Hybridization
assay (Q-PNA-FISH) (Cukusic et al., 2014). Telomerase
activity can be measured for example by using a
Telomere Repeat Amplification Protocol (TRAP) (Zhou
and Xing, 2012) (Table 1). Recently, several studies have
been focused on the expression of lubricin, also known
as proteoglycan 4 (PRG4) or superficial zone protein
(SZP), in different experimental conditions, in particular
in conjunction with physical activity (Musumeci et al.,
2013d). Lubricin is a chondroprotective glycoprotein
that serves as a critical boundary lubricant between
opposing cartilage surfaces (Musumeci et al., 2013e;
Leonardi et al., 2011). It has a major protective role in
8
Age-related degeneration of articular cartilage
Table 1. An overview of the key markers for the senescent chondrocytes and their functions.
CHONDROCYTE SENESCENCE MARKERS
Senescence markers Function
SA-βgal Hydrolase enzyme that catalyzes the hydrolysis of β-galactosides into monosaccharides only in senescent cells.
SAHF Packaging of proliferation-promoting genes in cellular senescence.
p53 Transcription factor involved in cell-cycle control, DNA repair, apoptosis, cellular stress responses and modulation of cellular senescence.
p21 Universal inhibitor of the cyclin-dependent kinases 1 required to arrest cells at the G1 and G2 checkpoints of the cell cycle after DNA damage.
pRb Potent repressor of genes that functions during DNA replication and thus causes cell cycle arrest.
p16 Tumor suppressor protein that plays an important role in cell cycle regulation by decelerating cell progression from G1 phase to S phase.
Caveolin 1 Protein that induces p38 MAPK activation and impairs the ability of chondrocytes to produce type II collagen and aggrecan.
Telomere length Telomere length shortens with each cell replication and characterizes cellular senescence.
Telomerase activity Telomerase activity diminishes with age and loses its ability to stabilize the shortened telomeres.
p
reventing cartilage wear and synovial cell adhesion,
proliferation, and in reducing the coefficient of friction
of the articular cartilage surface (Musumeci et al., 2013f;
Leonardi et al., 2012a,b). Since lubricin has a
fundamental role in maintaining the homeostasis of the
articular cartilage and in preventing its degeneration, we
hypothesized that its expression would decrease in
senescent chondrocytes and that it could be evaluated as
a new specific chondrocyte senescence marker. These
data have been confirmed in our recent and interesting
study in which we demonstrated the decreased
expression of lubricin is associated with chondrocytes
senescence as well as with OA (Musumeci et al., 2014d).
Conclusions
Although the direct relationship between the aging
process and the development of OA is not completely
understood, we may surmise that chondrocyte
senescence contributes to cartilage degeneration by
impairing the ability of these cells to maintain and repair
the cartilage tissue. Moreover, we have also seen that
these two processes (aging and OA) could be inter-
dependent. There are several lines of evidence that
suggest chondrocytes exposed to the osteoarthritic
environment” are characterized by oxidative stress and
production of cytokines, and this induces the so-called
stress-induced senescent state, which may contribute to
cartilage degeneration as we have discussed above. All
these observations suggest that a better understanding of
the changes arising with age in articular cartilage and
how they influence the response of the tissue to different
stressors, as well as the identification of new
increasingly sensitive senescence markers, would be
very useful in the preventive detection of the disease and
in its consequent treatment. Further research is required
to unravel the detailed mechanisms of senescence related
to the pathogenesis of OA. Strategies aimed at
preventing chondrocyte senescence could have a positive
impact on the development of new therapies for OA and
on halting the progression of this severe disease.
Acknowledgments. The study was funded by the Department of Bio-
Medical Sciences, University of Catania. The authors would like to thank
Prof. Iain Halliday for commenting and making corrections to the paper.
Conflict of Interest. The authors have no conflict of interest.
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Accepted July 10, 2014
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Age-related degeneration of articular cartilage
... As discussed earlier, telomeres shorten with the chronological age of chondrocyte donors and telomere changes are associated with senescence-like phenotypic drift (Martin and Buckwalter, 2001;Musumeci et al., 2015). Owing to the postmitotic nature of articular cartilage where chondrocyte renewal is virtually absent, stress-induced shortening of telomere is more likely than replicative shortening of telomeres. ...
... Stimuli including excessive mechanical loading, inflammation, and persistent oxidative stress cause an increased level of ROS which leads to DNA, protein, lipid and organelle damage. DNA damage induces telomere shortening that impacts the Hayflick limit, i.e., the ability of cells to re-enter the cell cycle for further rounds of cell division ultimately leads to cellular senescence and that propagation of senescence leads to cell death (Musumeci et al., 2015). Senescent chondrocytes arrest in the G1 phase of the cell cycle secret SASP, in which accumulation of the SASP-expressing cells contributes to tissue senescence by impairing the ECM attributed to the increased production of degradative enzymes, MMPs. ...
... Senescent chondrocytes arrest in the G1 phase of the cell cycle secret SASP, in which accumulation of the SASP-expressing cells contributes to tissue senescence by impairing the ECM attributed to the increased production of degradative enzymes, MMPs. Moreover, aging and/or OA-related decline in the anabolic and proliferative response to growth factors as well as the loss of cellularity support the concept that chondrocyte senescence contributes to the progression of cartilage degeneration (Musumeci et al., 2015). Apart from biological aging, in vitro serial expansion (four passages) of chondrocytes in monolayer culture reported to turn on the senescence-and dedifferentiation-mediated genes, leading to the loss of cartilage regeneration ability (Ashraf et al., 2016). ...
Full-text available
Article
Osteoarthritis (OA) is a joint degenerative disease that is an exceedingly common problem associated with aging. Aging is the principal risk factor for OA, but damage-related physiopathology of articular chondrocytes probably drives the mechanisms of joint degeneration by a progressive decline in the homeostatic and regenerative capacity of cells. Cellular aging is the manifestation of a complex interplay of cellular and molecular pathways underpinned by transcriptional, translational, and epigenetic mechanisms and niche factors, and unraveling this complexity will improve our understanding of underlying molecular changes that affect the ability of the articular cartilage to maintain or regenerate itself. This insight is imperative for developing new cell and drug therapies for OA disease that will target the specific causes of age-related functional decline. This review explores the key age-related changes within articular chondrocytes and discusses the molecular mechanisms that are commonly perturbed as cartilage ages and degenerates. Current efforts and emerging potential therapies in treating OA that are being employed to halt or decelerate the aging processes are also discussed.
... de l'élongation (ex. étendre les télomères par l'ajout de répétitions télomériques terminales[16]), qui présentent un intérêt particulier pour leur rôle dans les différents mécanismes et l'évolution (Figure 2)[15]. ...
... Raccourcissement des télomères. A chaque fois que les cellules se divisent, les télomères raccourcissent jusqu'à la "Limite de Hayflick"[15]. ...
Full-text available
Thesis
Single-stranded DNA binding proteins play an essential role in telomeres maintenance. Among these, we focused on the replication protein A (RPA) which has 6 single�stranded DNA binding domains (DBD) AF, and which binds and opens G-quadruplex structures (G4) present at the level of telomeric sequences rich in guanine. To understand the mechanism by which RPA unfolds telomeric G4s, we studied its interaction with oligonucleotides, which adopt G4, by targeting the different domains of RPA by OB-fold ligands. EMSA studies have shown that DBD-A/B domains are involved in the binding and opening activity of RPA to G4 (monomeric or multimeric). In addition, our results may suggest an allosteric effect of domain F on RPA binding and unfolding activity. Interestingly, the FRET studies have shown that RPA unfolded independently contiguous G4s and that opening kinetics depended on the stability of G4.
... At the same time, ADSCs treatment-induced cartilage repair can be reduced by miR-7-5p through transfection miR-7-5p into rat chondrocytes. Recent study suggested that in early stages of OA, autophagy could be protective for OA, autophagy levels of cartilage attenuated in the OA procession, especially in in middle and late stages of OA [33,[47][48][49]. ADSCs treatment could inverse an decreased cartilage autophagy levels, shown by the activation of autophagy markers (LC3A/B and p62) in OA rat [34], similar to our ndings. ...
Full-text available
Preprint
Background: Osteoarthritis (OA) is a highly degenerative joint disease, mainly companying with progressive destruction of articular cartilage. Adipose-derived stromal cells (ADSCs) therapy enhances articular cartilage repair, extracellular matrix (ECM) synthesis and attenuates joints inflammation, but specific mechanisms of therapeutic benefit remain poorly understood. This study aimed to clarify the therapeutic effects and mechanisms of ADSCs on cartilage damage in keen joint of OA rat model. Methods: In vivo study, destabilization of the medial meniscus (DMM) and anterior cruciate ligament transection (ACLT) surgery-induced OA rats were treated with allogeneic ADSCs by intra-articular injections for 6 weeks. The protective effect of ADSCs in vivo was measured using Safranin O and fast green staining, immunofluorescence and western blot analysis. Meanwhile, the miRNA-7-5p (miR-7-5p) expression was assessed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The mechanism of increased autophagy with ADSCs addition through decreasing miR-7-5p was revealed using oligonucleotides, adenovirus in rat chondrocytes. The luciferase reporter assay were used to reveal the molecular role of miR-7-5p and autophagy related 4A (ATG4A). The substrate of mTORC1 pathway: (p-)p70S6 and (p-)S6 in OA models with ADSCs addition were detected by western blotting. Results: The ADSCs treatment repaired the articular cartilage and maintained chondrocytes ECM homeostasis through modulating chondrocytes autophagy in OA model, indicators of the change of autophagic proteins expression and autophagic flux. Meanwhile, the increased autophagy induced by ADSCs treatment was closely related to the decreased expression of host-derived miR-7-5p, negative modulator of OA progression. Functional genomics (overexpression of genes) in vitro studies demonstrates inhibition of host-derived miR-7-5p in mediating the benefit of ADSCs administration in OA model. Then ATG4A were defined as a target gene of miR-7-5p, and the negative relation between miR-7-5p and ATG4A were investigated in OA model treated with ADSCs. Furthermore, miR-7-5p mediated chondrocyte autophagy by targeting ATG4A in OA cell model treated with ADSCs was confirmed with the rescue trial of ATG4A/miR-7-5p overexpression on rat chondrocyte. Finally, the mTORC1 signaling pathways mediated by host-derived miR-7-5p with ADSCs treatment were decreased in OA rats. Conclusions: ADSCs promotes the chondrocytes autophagy through decreasing miR-7-5p in articular cartilage by targeting ATG4A and a potential role for ADSCs based therapeutics for prevention of articular cartilage destruction and extracellular matrix (ECM) degradation in OA.
... The natural process of aging has an impact on articular cartilage with chondrocyte loss and a decline in metabolic response, alterations to the matrix and synovial tissue composition, and impairing the ability to maintain and repair these tissues [1]. Chondrocyte senescence contributes to cartilage degeneration, characterized by oxidative stress and the production of cytokines causing the so-called stress-induced senescent state [2]. A sedentary lifestyle with the consequent absence of loading for the cartilage accelerates the progression of cartilage degeneration [3]. ...
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Article
(1) Background: Cartilage degeneration with the natural aging process and the role of physical activity on cartilage wellness is still not clear. The objective of the present review was to understand how different physical activity interventions affect the cartilage and to propose a Standard Operating Procedure for an exercise program to maintain knee joint health; (2) Methods: Articles were collected on three different electronic databases and screened against the eligibility criteria. Results were collected in tables and the main outcomes were discussed narratively; (3) Results: A total of 24 studies have been included after the screening process and aerobic, strength, flexibility, postural balance, and mobility interventions were detected. Different protocols and types of interventions were adopted by the authors; (4) Conclusions: Physical activity interventions have mainly positive outcomes on cartilage structure, but the protocols adopted are different and various. A Standard Operating Procedure has been proposed for a physical intervention focalized on cartilage wellness that could be adopted as an intervention in the clinical setting. Furthermore, the creation of a standardized protocol wants to help scientific research to move in the same direction.
... Autophagy, also known as type II programmed cell death and non-apoptotic cell death program (13), plays an important role in the progression of KOA (14). It is an intracellular reaction that can transform senescent and damaged organelles and proteins into substances needed by the cells and thus, autophagy is thought to exert a cartilage protective effect in KOA (15). ...
Full-text available
Article
Background: Knee osteoarthritis (KOA) is a leading cause of chronic pain and disability, and as such, it poses a significant economic burden. Traditional Chinese medicine (TCM), as well as complementary and alternative medicine, can offer safe and effective treatments for KOA. Cangxitongbi (CXTB) capsule is a Chinese patented medicine for KOA treatment and has a remarkable curative effect. This article evaluated the effects and mechanisms of CXTB in protecting joint cartilage in vivo. Methods: The KOA model was constructed in rats using the modified Hulth method. CXTB (35 mg/kg) was administered intragastrically for 4 weeks. Hematoxylin and eosin (HE) staining of the knee articular were performed to evaluate the efficiency of CXTB. Western blot analysis, quantitative polymerase chain reaction (qPCR), and enzyme-linked immunosorbent assay (ELISA) were used to investigate the protective mechanisms of CXTB in joint cartilage. Results: CXTB effectively improved the morphological structure of the cartilage and bone in the knee joint by enhancing autophagy and regulating the expression of related protease and inflammatory factors. Furthermore, CXTB downregulated the expression of the long non-coding RNA (lncRNA) Hox transcript antisense intergenic RNA (HOTAIR) and inhibited the activation of the p38MAPK pathway. Conversely, overexpression of lncRNA HOTAIR suppressed the protective effects of CXTB on the knee joint. Conclusions: CXTB capsules can protect the knee articular cartilage in rats through the lncRNA HOTAIR/p38MAPK pathway.
... an enhanced understanding of the mechanisms relevant to oa development is critical for the investigation of novel treatment strategies. a well-established hypothesis is that chondrocyte degeneration causes premature aging due to excessive mechanical load or oxidative stress, leading to stress-induced aging, and ultimately, the onset of oa (19). Therefore, insights into the underlying mechanisms of chondrocyte degeneration are critical to researching oa development. ...
Full-text available
Article
Osteoarthritis (OA) is one of the most prevalent pain‑inducing and disabling diseases globally. Aging is a primary contributing factor to the progression of OA. Forkhead box protein O4 (FOXO4) is known to be involved in the cell cycle and apoptosis regulation. The aim of the present study was to investigate the association between FOXO4 expression and chondrocyte degeneration in rats. Chondrocytes were assigned to the control (4‑week‑old rats), natural degeneration (16‑week‑old rats) or induced degeneration (IL‑1β‑treated chondrocytes from 4‑week‑old rats) groups. Immunocytochemical analysis with β‑galactosidase staining revealed a greater number of stained cells present in the natural and induced degeneration groups than in the control group. PCR analysis indicated lower mRNA expression levels of collagen type II α1 chain (Col2α) and higher levels of FOXO4, and western blotting revealed reduced Col2α protein expression levels and significantly elevated FOXO4 levels in the natural and induced degeneration groups, compared with those in the control group. The results of the present study revealed that FOXO4 expression was altered in the natural and induced degeneration groups, and further research and exploration are needed to clarify the underlying mechanism.
... The risk factors of OA consists of combination of local biomechanical (muscle strength, specific bone/joint shapes, joint loads and alignment, joint overload and joint injury), into personlevel factors (sociodemographic characteristics, obesity, genetic predispositions, bone density and mass, sedentary lifestyle, diet-related factors) and metabolic disorders factors. There is growing evidence of the association between OA and metabolic syndrome rather than obesity itself has the greatest impact on the initiation and progression of disease severity (15)(16)(17)(18)(19)(20)(21)(22)(23). Traditionally OA has been considered a degenerative "tear and wear" disease leading to loss of cartilage, but today this view changed. ...
Article
Objective: Osteoarthritis (OA) results from loss of cartilage in-tegrity in association with changes to the structure of the entire joint. Treatment of OA is based on different pharmaceutical and no phar-maceutical approaches and the latter include the use of spa-therapy. The biological effects of mud-bath therapy are mainly secondary to heat stimulation and to physic-chemical properties of mineral waters and mud-packs. Mud-bath therapy likely exerts its effects modulating several cytokines and other molecules involved in inflammation and cartilage degradation. Our aim was to perform an updated meta-analysis of the effectiveness of the mud-bath therapy on knee osteoarthritis and briefly to discuss the mechanisms of action of this treatment. Materials and methods: A MEDLINE on PubMed for articles on knee OA and spa therapy published from 1995 through up to April 2019 was performed. Then, we checked the Cochrane Central Register of Controlled Trials to find additional references included up to April 2019. Articles were included if in accordance with the eligibility cri-teria. Sample size and effect sizes were processed with the MedCalc software package. Results: Twenty one studies met the inclusion criteria and were included in meta-analysis. We examined WOMAC Index and VAS pain. We found significant improvements in function scores and painful symptoms after mud-bath therapy in patients with knee joint osteoarthritis. Conclusions: Spa therapy is a non-drug treatment modalities, non invasive, complication-free, and cost-effective alternative modality for the conservative treatment of knee osteoarthritis. It cannot substitute for conventional therapy but can integrated or alternated to it. Treatment with mud-bath therapy may relieve pain, stiffness and improve functio-nal status in patients with knee OA.