Senolytic activity of small molecular polyphenols from olive restores chondrocyte redifferentiation and promotes a pro-regenerative environment in ...
←
→
Page content transcription
If your browser does not render page correctly, please read the page content below
www.aging-us.com AGING 2020, Vol. 12, No. 16
Priority Research Paper
Senolytic activity of small molecular polyphenols from olive restores
chondrocyte redifferentiation and promotes a pro-regenerative
environment in osteoarthritis
Marta Varela-Eirín1, Paula Carpintero-Fernández1, Agustín Sánchez-Temprano1, Adrián Varela-
Vázquez1, Carlos Luis Paíno2, Antonio Casado-Díaz3, Alfonso Calañas Continente3, Virginia Mato4,
Eduardo Fonseca1, Mustapha Kandouz5, Alfonso Blanco6, José Ramón Caeiro7, María D. Mayán1
1
CellCOM Research Group, Instituto de Investigación Biomédica de A Coruña (INIBIC), Servizo Galego de Saúde
(SERGAS), Universidade da Coruña (UDC), A Coruña, Spain
2
Neurobiology-Research Service, Hospital Universitario Ramón y Cajal (IRYCIS), Madrid, Spain
3
UGC Endocrinology and Nutrition, Maimónides Biomedical Research Institute of Córdoba (IMIBIC), Hospital
Universitario Reina Sofía – CIBERFES, Universidad de Córdoba, Córdoba, Spain
4
Centre for Medical Informatics and Radiological Diagnosis, Universidade da Coruña, A Coruña, Spain
5
Department of Pathology, School of Medicine, Wayne State University, Detroit, MI 48202, USA
6
Flow Cytometry Core Technologies, UCD Conway Institute, University College Dublin, Dublin, Ireland
7
Department of Orthopaedic Surgery and Traumatology, Complexo Hospitalario Universitario de Santiago de
Compostela (CHUS), Universidade de Santiago de Compostela (USC), Choupana s/n, Santiago de Compostela,
Spain
Correspondence to: María D. Mayán; email: Ma.Dolores.Mayan.Santos@sergas.es
Keywords: senescence, dedifferentiation, osteoarthritis, connexin43, tissue regeneration
Received: March 18, 2020 Accepted: July 13, 2020 Published: August 3, 2020
Copyright: Varela-Eirín et al. This is an open-access article distributed under the terms of the Creative Commons Attribution
License (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original
author and source are credited.
ABSTRACT
Articular cartilage and synovial tissue from patients with osteoarthritis (OA) show an overactivity of connexin43
(Cx43) and accumulation of senescent cells associated with disrupted tissue regeneration and disease
progression. The aim of this study was to determine the effect of oleuropein on Cx43 and cellular senescence
for tissue engineering and regenerative medicine strategies for OA treatment. Oleuropein regulates Cx43
promoter activity and enhances the propensity of hMSCs to differentiate into chondrocytes and bone cells,
reducing adipogenesis. This small molecule reduce Cx43 levels and decrease Twist-1 activity in osteoarthritic
chondrocytes (OACs), leading to redifferentiation, restoring the synthesis of cartilage ECM components (Col2A1
and proteoglycans), and reducing the inflammatory and catabolic factors mediated by NF-kB (IL-1ß, IL-6, COX-2
and MMP-3), in addition to lowering cellular senescence in OACs, synovial and bone cells. Our in vitro results
demonstrate the use of olive-derived polyphenols, such as oleuropein, as potentially effective therapeutic
agents to improve chondrogenesis of hMSCs, to induce chondrocyte re-differentiation in OACs and clearing out
senescent cells in joint tissues in order to prevent or stop the progression of the disease.
INTRODUCTION senescent cells [1–4] together with increased
inflammation and breakdown of cartilage extracellular
Articular cartilage from patients with osteoarthritis matrix (ECM) [5]. Importantly, cartilage and synovial
(OA) shows accumulation of dedifferentiated and tissue from OA patients contain high levels of the gap
www.aging-us.com 15882 AGING
junction protein connexin43 (Cx43). Targeting Cx43 downregulation of Cx43 reduces stemness and
might be a promising approach to treat several age- accumulation of senescent cells, indicating that Cx43
related and chronic degenerative diseases by modulating acts as a molecular switch in the phenotype of
tissue regeneration, inflammation and response to injury chondrocyte within a wound healing process [2]. In fact,
[6]. Cx43 belongs to the integral membrane protein downregulation of Cx43 in different wound healing and
family called connexins, that enable direct age-related disorders halts disease progression by
communication between neighboring cells via restoring tissue regeneration [45–47].
hemichannels, gap junctions, extracellular vesicles and
tunneling nanotubes [7]. Additionally, connexins act as Oleuropein is the most abundant polyphenol in the
signaling hubs regulating different signaling pathways leaves and fruit of the olive plant and is a potent
via their cytoplasmic domains [2, 8]. Cx43 is the major antioxidant agent with anti-tumour and anti-
Cx protein expressed in chondrocytes, synovial cells inflammatory properties [48, 49]. The mechanism of
(SC) and bone cells (BC) [9–11] and it has been action of this polyphenol is under investigation [48].
involved in normal development and function of joint Some studies showed that oleuropein and its major
tissues [12, 13] and joint disorders [9–11, 14, 15] metabolite hydroxytyrosol have antioxidant activity by
inhibition and/or scavenging of ROS [50], which can
During tissue regeneration and following injury, reduce NF-kB activation [51, 52]. Other mechanistic
dedifferentiation, redifferentiation and senescence studies implicate nitric oxide (NO) production [53, 54]
processes play finely tuned temporal and spatial roles to or autophagy and inhibition of the mammalian target of
reverse the loss [2, 16]. In addition, accumulation of rapamycin (mTOR) [55, 56]. A gene expression
senescent cells is described to play a major role in OA profiling study has suggested that oleuropein affects the
progression [1, 17–22]. Cellular senescence is a stable expression of genes involved in oxidative stress,
cell-cycle arrest with increased expression of cell cycle inflammation, fibrosis, cell proliferation or
inhibitors such as p16INK4A and enhanced synthesis of differentiation [57], suggesting that the beneficial
the senescence-associated secretory phenotype (SASP) effects of this molecule may be multifactorial and
factors, which mainly consist of inflammatory cytokines context-dependent [57]. Innovative approaches based on
and ECM degrading enzymes, among other factors [23]. functional foods have been recently studied and many
The elimination of senescent cells using senolytics has beneficial effects of dietary olive oil on human health
been described to attenuate cartilage and joint were already described [58–62]. In fact, extra virgin
degeneration in OA [1, 24–26]. In fact, some of these olive oil and olive leaf extract supplemented diets have
drugs including UBX0101 and the natural-occurring been shown to reduce inflammation and preserve
flavone Fisetin [27, 28] with potential senolytic activity cartilage, muscle and joint function in rat preclinical
are currently under clinical trial for OA treatment models of OA [63–68].
(NCT04210986; NCT04349956; NCT04229225;
NCT04129944) [29–32]. In this study, we have used a small-scale screening to
identify compounds that downregulate Cx43. Here we
Cx43 has been involved in different phases of tissue describe the use of small molecules based on olive
regeneration including chronic inflammation, cell phenolic compounds to downregulate Cx43 in OA by
differentiation and cellular senescence [33, 34]. using 2D and 3D human cartilage models. We have
Overexpression of Cx43 and enhanced gap junction found that oleuropein decreases Cx43 promoter activity
intercellular communication (GJIC) in osteoarthritic and GJIC, thus enhancing osteogenesis and
chondrocytes (OACs) compromise their ability to re- chondrogenesis in hMSCs and redifferentiation of
differentiate, promoting a stem-like state by activating OACs. Beside downregulation of Cx43, olive-derived
Twist-1, which leads to dedifferentiation via epithelial- small polyphenols reduce cellular senescence in OACs,
to-mesenchymal transition (EMT) [2]. However, we BC and SC, in addition to inflammatory and catabolic
have also previously observed that high levels of Cx43 activities related to cartilage degradation in OA patients.
lead to p53/p21-mediated cellular senescence [2].
Increased levels of Cx43 in OA cartilage is extensively RESULTS
described in the literature [9, 11, 35–37], as well as the
presence of dedifferentiation (stem-like state) and Olive-derived polyphenols impair adipogenesis and
proliferative chondrocytes [4, 38–44] together with the enhance the chondrogenic and osteogenic ability of
accumulation of senescent cells [1, 18, 20, 29], hMSCs
indicating that both phenotypes, dedifferentiated and
senescent chondrocytes, co-exist in cartilage from OA We used a small-scale screening to identify
patients and contribute to the progression of the disease. compounds that downregulate Cx43 (data not shown),
However, we have recently demonstrated that and we identified the small molecule oleuropein
www.aging-us.com 15883 AGING
(Figure 1A). Oleuropein and an olive-extract (OE) Downregulation of Cx43 activity by oleuropein
significantly reduced Cx43 protein levels in OACs downregulates Twist-1 and enhances
(Figure 1A). MTT assays showed no effect of 0.1-10 redifferentiation of OACs
µM oleuropein on cell viability of primary
chondrocytes and hMSCs (Supplementary Figure 1). Oleuropein modulation of Cx43 significantly reduced
Based on these results and other studies [69], 10 µM GJIC in OACs (Figure 2A), but not in healthy
was selected as the highest concentration with no toxic chondrocytes (Figure 2B). The decrease in Cx43 and
effect after 17 h in culture. GJIC was correlated with a significant reduction in the
levels of the stemness markers CD105 and CD166
We next examined hMSCs differentiation capacity in the (Figure 2C). This result was consistent with our
presence of oleuropein (Figure 1B, 1C and Supplementary previous observations, where CD105 and CD166 were
Figure 2). In accordance with previous results [69], reduced when Cx43 was downregulated or when OACs
oleuropein-treated hMSCs showed significantly less were redifferentiated. In fact, oleuropein effectively
adipogenic differentiation ability, whereas osteogenesis improved the OACs phenotype, detected by the increase
was significantly increased (Figure 1B and of Col2A1 levels and the decrease of proinflammatory
Supplementary Figure 2A, 2B). Furthermore, a 3D mediators and MMP-3 levels (Figure 2D, 2E).
micromass culture system using chondrogenic medium Oleuropein treatment reduced overall Cx43 positivity,
supplemented with 10 µM oleuropein or OE revealed increased Col2A1 levels (Figure 2D) and decreased
increased ECM properties, reflecting a greater degree of interleukin 6 (IL-6), COX-2, IL-1ß and MMP-3 gene
chondrogenic differentiation, with higher levels of expression (Figure 2E) and protein levels (Figure 2F).
Col2A1 deposition and increased levels of aggrecan
(ACAN) gene expression (Figure 1C and Supplementary Next, we sought to confirm whether oleuropein would
Figure 2C). Remarkably, decreased Cx43 gene expression target chondrocyte plasticity in 3D cultures. Treatment
was detected in hMSCs during differentiation, mainly of OACs grown as a 3D culture in chondrogenic
during chondrogenesis (Figure 1D, 1E and Supplementary medium with oleuropein increased the deposition of
Figure 3A). We have also observed changes in Cx43 proteoglycans and Col2A1 (Figure 3A), improving the
phosphorylation pattern at 7 and 14 days of chondrogenic ECM structure by decreasing Cx43 levels (Figure 3B).
differentiation (Figure 1D), which can affect Cx43
stability and channel activity. Different Cx43 To explore the effect of oleuropein on cell plasticity,
phosphorylation patterns were also detected during OACs were grown in adipogenic medium supplemented
adipogenesis and osteogenesis, suggesting differential with oleuropein, which significantly decreased their
regulation of Cx43 during hMSCs differentiation adipogenic differentiation (Figure 3C). However,
(Supplementary Figure 3B). The treatment of hMSCs oleuropein promoted osteogenesis when OACs were
during chondrogenic differentiation with oleuropein for grown in osteogenic medium supplemented with 10 µM
14 days caused an additional 1.7 fold reduction in Cx43 of oleuropein (Supplementary Figure 5).
gene expression (Figure 1E).
Upregulation of Cx43 in OA involves dedifferentiation
In concordance with these results, OACs’ via chondrocyte-to-mesenchymal transition by Twist-1
redifferentiation for 7 days with oleuropein decreased activation, which was also reported in OA cartilage [2,
Cx43 protein levels, but it did not affect its 70, 71]. OACs were treated with the arthritic insult
phosphorylation pattern (Figure 1F). However, we did oligomycin, which induces cellular ROS production and
not observe changes in the phosphorylation pattern cartilage degradation [72, 73]. After oleuropein
when hMSCs or chondrocytes were treated with treatment, OACs showed a significant reduction in the
oleuropein (Figure 1F and Supplementary Figure 4A). nuclear localization of Twist-1 (Figure 3D). Nuclear
localization of Twist-1 in the presence of oligomycin
It is important to note that these results were obtained was attenuated when Cx43 protein levels were reduced
during differentiation of hMSCs under osteogenesis or by oleuropein treatment of OACs (Figure 3E). In
chondrogenesis (Figure 1B–1E) and dedifferentiated addition, oleuropein treatment in OACs reduced the
OACs in normal and in chondrogenic medium (Figure expression of the mesenchymal and EMT markers N-
1A and 1F). However, the treatment of undifferentiated cadherin and vimentin (Figure 3F).
hMSCs cultured in basal growth medium with
oleuropein/OE increased Cx43 levels (Supplementary Oleuropein modulates Cx43 gene promoter activity
Figure 4A and 4B) and GJIC (Supplementary Figure
4C), indicating that the effect of these olive derived In chondrocytes, decreased Cx43 protein levels were
polyphenols may be different depending on the cellular detected when OACs and the T/C-28a2 cell line were
context. treated with oleuropein (Figures 1A and 4A). However,
www.aging-us.com 15884 AGING
Figure 1. Downregulation of Cx43 during chondrogenesis improves differentiation towards chondrocytes. (A) Treatment of OACs with oleuropein (Oleu) or olive extract (OE) for 2 h significantly downregulates Cx43 protein detected by western-blot and flow cytometry. Median fluorescence intensity (MFI) ratios of oleuropein and OE treatments with respect to their untreated controls of each experiment are represented (n=10 independent experiments, P=0.0003). (B) Differentiation capacity of hMSCs isolated from bone marrow grown in adipogenic (top, 21 days) or osteogenic (bottom, 21 days) medium supplemented with 10 µM oleuropein or 10 µM OE. hMSCs cultured in growth medium were used as a control. Top, adipogenic evaluation by oil red O for lipid staining and by PPARγ gene expression. Data represent the ratio of cells containing lipid deposits to the total number of cells (n=3 independent experiments, P
Figure 2. Downregulation of Cx43 by oleuropein decreases GJIC and improves the phenotype of OACs. (A) Oleuropein (Oleu) treatment significantly decreases GJIC evaluated by an SL/DT assay when OACs were exposed with this molecule for 2 h (top, n=6 independent experiments; Student’s t test, P
these changes were not evident in treated Cx43- for 1 h with oleuropein and the mitochondrial inhibitor overexpressing T/C-28a2 chondrocytes (Figure 4A, oligomycin, which enhanced Cx43 gene expression bottom), suggesting that oleuropein may affect Cx43 (Figures 3E, 4B). Cx43 promoter activity decreased gene promoter activity rather than protein stability. We after oleuropein treatment (Figure 4B), while thus measured whether oleuropein affected the activity oligomycin increased the luminescence signal, and the of the Cx43 gene promoter using a real-time reporter effect of oligomycin was significantly attenuated in the system. T/C-28a2 chondrocytes were transfected with a presence of oleuropein (Figure 4B), which was firefly luciferase reporter vector containing the correlated with Cx43 gene expression (Figure 4B). regulatory regions of the Cx43 promoter and incubated Luminescence signal strongly correlated with the effects Figure 3. Oleuropein treatment enhances chondrocyte redifferentiation. (A) Immunohistochemistry of Col2A1 (4-6 independent experiments; one-way ANOVA, P=0.0019) and toluidine blue staining of proteoglycan subunits (n=6 independent experiments; one-way ANOVA, P=0.059) indicate significant enrichment in ECM components in OACs micromasses grown in 3D culture for 30 days in chondrogenic medium (CM) when supplemented with 10 µM oleuropein (Oleu) or OE. (B) Cx43 protein levels detected by western blot (and normalized to Ponceau staining) are reduced when OACs micromasses are exposed to CM supplemented with 10 µM oleuropein or OE for 21 days (n=3 independent experiments; one-way ANOVA, P=0.0328). (C) Oil red staining showing reduced OACs dedifferentiation upon exposure to Oleu or OE in adipogenic medium (n=5 independent experiments; one-way ANOVA, P=0.0001). (D) Nuclear levels of Twist-1 were decreased in OACs cultured with 10 µM oleuropein for 2 h. Lamin A was used as a loading control (n=3 independent experiments; Student’s t test, P=0.001). (E) Cx43 protein levels in primary OACs after 1-h treatment with oleuropein or oligomycin. Western blot represents n=4 independent experiments. Quantification is shown on the right (one-way ANOVA, P=0.0036). On the right, immunofluorescence for Twist-1 (red) in primary OACs treated with 5 µg/ml oligomycin and 10 µM oleuropein for 1 h. The graph represents the percentage of cells with Twist-1 nuclear localization (n=4 independent experiments; one-way ANOVA, P=0.0067). (F) The mRNA expression of the EMT markers Twist- 1, N-Cadherin and Vimentin in OACs treated with 10 µM oleuropein for 2 h. Data were normalized to HPRT-1 levels. n= 5 independent experiments; Student’s t test: P
of oleuropein and oligomycin on protein levels (Figures Cx43 upregulation due to the oligomycin insult
3E and 4C). These results indicate that oleuropein significantly contributed to increase cellular senescence
affects Cx43 gene promoter activity, thus in OACs accumulated after 5 days in monolayer (Figure
downregulating Cx43 protein levels (Figure 1A and 5B, left), whereas co-treatment with oleuropein
Figure 4A–4C) and GJIC in OACs (Figure 2B). significantly halted the accumulation of senescent cells
after 24 h (Figure 5B, left) and 7 days under treatment
Oleuropein enhances elimination of senescent cells (Figure 5B, right). Interestingly, increased SA-βGal
activity was detected when the T/C-28a2 chondrocytes
Senescent cells through their secretory activity (SASP) were treated with bleomycin to induce cellular
can promote dedifferentiation and reprogramming in senescence for 24 h, while the exposure to oleuropein
neighboring cells in the context of tissue injury [74]. for 24 h reduced the number of senescent cells
OACs treated with 10 µM oleuropein for 7/14 days in (Supplementary Figure 6B). In accordance with these
growth medium showed a significant reduction of results, treatment of OACs with oleuropein led to
senescent cells accumulated after 5 days of primary decreased levels of the senescence biomarkers p16INK4A
culture (Figure 5A). Consistent with these models, (Figure 5C) and p53/p21 (Figure 5D). Furthermore, the
Figure 4. Oleuropein modulates the Cx43 promoter activity in chondrocytes. (A) Treatment with 10 µM oleuropein for 2 h
decreases Cx43 protein levels in T/C-28a2 cells (n=4 independent experiments, Student’s t test, P=0.0012), but this effect was not observed
in the same cell line overexpressing Cx43 (pIRES-Cx43)(n=3 independent experiments, Student’s t test, P=0.0624). (B) Luciferase reporter
assay indicating that oleuropein inhibits Cx43 promoter activity. The graphs indicate the normalized luminescence activity in the T/C-28a2
transfected with a pGL3-basic plasmid containing 300 base pairs of Cx43 promoter ligated to the luciferase gene. Cells were cultured in
DMEM with 10% FBS (UT) and with 5 µg/ml oligomycin or 10 µM oleuropein for 1 h as indicated (n=4 independent experiments; one-way
ANOVA, P=0.0012). On the right, Cx43 gene expression under 5 µg/ml oligomycin and 10 µM oleuropein treatment in OACs treated for 1 h
(n=4 independent experiments; one-way ANOVA, P=0.0002). Data were normalized to HPRT-1 levels. (C) Immunofluorescence assays of Cx43
in OACs treated with 10 µM oleuropein or 5 µg/ml oligomycin for 1 h. Data were normalized to the untreated condition (n=3 independent
experiments; one-way ANOVA, P
proliferative arrest observed after the treatment of T/C- of hMSCs to oleuropein and OE may have potential
28a2 healthy chondrocytes with the Cyclin-Dependent applications in preventive and regenerative medicine in
Kinase 4/6 inhibitor palbociclib to induce senescence other bone and cartilage disorders.
was partially inhibited by the co-treatment with
oleuropein (Figure 5E). Oleuropein reduced the Our data show decreased Cx43 and GJIC levels in the
accumulation of senescent cells and attenuated the presence of oleuropein in OACs and in differentiating
oligomycin-induced SASP secretion detected by IL-6, hMSCs, but increased Cx43 and GJIC levels in
COX-2 and IL-1ß gene expression in chondrocytes undifferentiated hMSCs, suggesting that the effect of this
(Figure 5F). The SASP, including IL-6 gene expression, molecule on GJIC depends on its effect on Cx43 levels
can be activated by NF-κB [75]. Oleuropein protected (or subcellular localization). In fact, phosphorylation of
from the increase of Cx43 under TNFα treatment Cx43 affects protein stability and GJIC activity [79] and
(Figure 5G, left), and NF-κB (p65) activation by TNFα we have detected changes in Cx43 phosphorylation
in OACs was diminished when cells were exposed to pattern during hMSCs differentiation but not under
oleuropein for 1 h (Figure 5G). Further, NF-κB nuclear oleuropein treatment indicating that oleuropein affects
translocation was partially abolished in OACs treated Cx43 levels more than gap junction plaque activity or
with oleuropein for only 2 h (Figure 5H). modulation. We cannot therefore discard that the effect
of oleuropein may also depend on channel-independent
To further test the senolytic activity of oleuropein, SC activities, which involve the signaling hub’s ability of
and BC from OA patients were treated with oleuropein Cx43 to recruit proteins to the membrane [34, 80, 81] or
(Figure 6). We observed decreased Cx43 protein levels its ability to control gene transcription [82]. It is
after oleuropein exposure (Figure 6A and 6D), together important to note that oleuropein may have other targets
with a significant reduction in senescent cells that may contribute to the drug effect. Despite this
accumulated after 5 days in primary culture (Figure 6B limitation and based on our results, we expect that the
and 6E), confirmed by p16INK4A gene expression and the effect of oleuropein occurs at least partially through Cx43
synthesis of the SASP factors IL-1ß, COX-2 and IL-6 modulation. Here, we show that oleuropein restores
(Figure 6C and 6F). chondrocyte phenotype detected by reduced levels of the
stem-markers CD105, CD166, N-cad and vimentin
DISCUSSION (Figure 2C, 3F). The effect of oleuropein in chondrocyte
plasticity correlated with activation of redifferentiation
Previous data from our group demonstrate that Cx43 via downregulation of Cx43 and Twist-1 (Figure 3D–3F),
downregulation improves the chondrocyte phenotype, leading to increased levels of proteoglycans and Col2A1
protecting chondrocytes from dedifferentiation and together with decreased levels of inflammatory cytokines
senescence [2]. Although oleuropein and olive-based and metalloproteinases (Figures 2D–2G, and 3A).
diets were reported to protect from OA progression [76,
77], there was no solid evidence about its underlying In cell culture and in cartilage, OACs undergo
molecular mechanisms. In our study, we show that dedifferentiation and senescence [2, 18, 20].
oleuropein modulates Cx43 gene promoter activity, Elimination of senescent cells in vivo using the
reducing Cx43 and GJIC in OACs. Indeed, our data senolytic drug UBX0101 has been demonstrated to
indicate that Cx43 downregulation by oleuropein in improve cartilage regeneration after articular joint
OACs improves cell phenotype by protecting injury in mice [1]. Here we show that oleuropein
chondrocytes from Twist-1 activation and from reduces cellular senescence in OACs, SC and BC and
accumulation of senescent cells (Figures 2, 3, 5 and 6). protects from accumulation of senescent cells under an
This is the first study that demonstrated one of the arthritic insult (Figures 5A, 5B, 6B and 6F). NF-kB has
potential mechanisms of oleuropein in OACs, SC and been shown to regulate the inflammatory components of
BC (Figures 5 and 6) from patients, and in hMSCs the SASP, together with other factors [83, 84]. In this
(Figure 1), with relevant applications in regenerative study the reduction of senescence is accompanied by
medicine. reduced NF-kB activity, and therefore reduced synthesis
of SASP (Figure 5F). Notably, these components
Our results show that the effects of oleuropein are often enhance inflammation, senescence and activate
equal or even smaller than those of the olive extract, dedifferentiation and cellular reprogramming of nearby
suggesting that other compounds may synergize with non-senescent cells (e.g. via IL-6) [74], contributing to
oleuropein activity in chondrocytes [78]. On the other the stem-like state of chondrocytes in OA and to the
hand, the treatment of hMSCs with oleuropein or OE accumulation of senescent cells. Accordingly, we have
during differentiation leads to downregulation of Cx43 previously reported that upregulation of Cx43 leads to
and GJIC, enhancing osteogenesis and chondrogenesis, p53/p16 upregulation and senescence [2]. Using the
but reducing adipogenesis. This differential sensitivity T/C-28a2 cells with a Cx43 overexpression vector and a
www.aging-us.com 15889 AGING
CRISPR/Cas9-mediated heterozygous Cx43 gene Cellular reprogramming, dedifferentiation via EMT and knockdown cell line we have demonstrated that Cx43 is senescence play active roles during tissue regeneration an upstream effector of both senescence (involving p53 [85, 86]. Accumulation of dedifferentiated (stem-like and p16 pathways) and NF-kB activation [2]. cells) and senescent cells leads to impaired tissue Figure 5. Cx43 downregulation by oleuropein decreased chondrocyte senescence. (A) SA-βGal activity detected by flow cytometry in OACs treated with 10 µM oleuropein (Oleu) for 7 and 14 days (n=3–7 independent experiments; one-way ANOVA, P
regeneration and fibrosis with loss of tissue function oleuropein, will potentially contribute to create a [87] (Figure 7). Understanding and manipulating the regeneration-permissive environment in OA patients. complex Cx43 signaling would expand our opportunities for modulating wound-healing related Our preliminary study is the first to demonstrate the disorders such as OA. So far, our results indicate that effect of this polyphenol on Cx43 activity and molecules that reduce Cx43 levels in OA, including senescence. Besides, modulation of Cx43 by oleuropein Figure 6. Oleuropein treatment decreased cellular senescence in synoviocytes and bone cells isolated from patients. (A) Cx43 protein levels analyzed by western blot in synoviocytes treated with 10 μM oleuropein for 2 h (n=7 independent experiments, P=0.0313). (B) Treatment of synoviocytes with 10 μM of oleuropein for 7 days detected by SA-βGal activity (n=4 independent experiments, P
shifts the hMSCs differentiation capacity towards oleuropein controls Cx43 gene expression and acts as a osteogenic and chondrogenic lineages, while decreasing senolytic drug that may serve as a potential agent to adipogenic differentiation. It is important to remark that improve both the effectiveness of stem cell therapy and aging has been reported to reduce the reparative cartilage and joint regeneration in patients to stop OA potential of MSCs [31]. Hence, the use of small progression. Thus, the implementation of therapies polyphenols such as oleuropein may be useful in cell based on Cx43 modulators or innovative approaches therapy approaches that aim to promote cartilage and based on functional foods enriched in olive extracts may bone regeneration, as the osteogenesis/adipogenesis improve joint functions and quality of life for OA switch has been associated with different bone disorders patients by reducing senescence and inducing a pro- [88, 89]. Altogether, these findings indicate that regenerative environment. Figure 7. Cx43 overactivity contributes to disease progression. Cx43 overexpression leads to accumulation of dedifferentiated and senescent cells involved in disease progression in OA patients. These phenotypic changes results in the synthesis of ECM remodeling factors involved in tissue degradation (MMPs) and proinflammatory factors, such as IL-1ß and IL-6, which facilitate the dedifferentiation and reprogramming of neighboring cells. These factors may further spread senescence and dedifferentiation to surrounding tissues contributing to joint degeneration. Downregulation of Cx43 by oleuropein treatment contributes to the elimination of senescent cells and redifferentiation of osteoarthritic chondrocytes into fully differentiated cells, able to support the ECM composition and restoring the regenerative capacity of the tissue. However, oleuropein may have other targets that may contribute to the drug effect. In addition, oleuropein treatment might improve the effectiveness of stem cell therapy, by promoting chondrogenic and osteogenic differentiation, and by inhibiting adipogenesis. www.aging-us.com 15892 AGING
MATERIALS AND METHODS containing 41.5% of oleuropein, donated by the Clinical
Management Unit of Endocrinology and Nutrition
Tissue collection, processing and cell culture (IMIBIC, Córdoba, Spain). OE was dissolved in culture
medium to a stock concentration equivalent to 100 µM
Collection and processing of cartilage from human oleuropein. Both compounds were dissolved in the cell
knees and femoral heads from adult donors undergoing culture medium and added to the cells for short-term
joint surgery were performed as previously described (1–2 h) or long-term (7–14—21 days; each 48 hours)
[10]. Briefly, primary chondrocytes were isolated from treatments. Bone cells and synoviocytes were treated
cartilage by mechanical (dicing) and enzymatic with oleuropein 10 µM for 2 h at a 70% confluence. For
digestion, with an incubation with 0.5 mg/mL of cell treatments, chondrocytes at a 70-80% confluence
trypsin-EDTA (Gibco, Thermo Fisher Scientific) for 10 were treated for 1 h with either 5 µg/ml oligomycin
min at 37ºC, followed by an overnight incubation at (Sigma-Aldrich) or 10 ng/mL TNFα (Immunotools). In
37ºC with a 2 mg/mL collagenase IV solution, with addition, these cells were also treated simultaneously
shaking. Finally, digested cartilage was filtered through for 1 h with the combination of oligomycin or TNFα
a 100 µm strainer and chondrocytes were seeded onto with 10 µM oleuropein. Cell proliferation arrest in T/C-
100 mm dish plates (Corning, Sigma-Aldrich). The 28a2 healthy chondrocytes was performed by a 24h-
study was conducted with the approval of the treatment with 10 µM of Palbociclib (APExBIO) or the
institutional ethics committee (C.0003333, 2012/094 & combination of Palbociclib and 10 µM oleuropein.
2015/029) after the acquisition of signed informed
consents. We have collected cartilage samples (1 Cell viability assay
sample per patient) from a total amount of 22 donors
(14 OA donors and 8 healthy donors) from hip and knee Cells at a 75% confluence were treated with 0.1 µM, 1
joints with a mean age of 72.1±4,7 years (50% women µM, 10 µM and 10 mM oleuropein for 17 h. Drug
and 50% men) within OA donors, and a mean age of cytotoxicity was evaluated by the colorimetric MTT
81.62±2.9 years (75% women and 25% men) within assay (Cell Proliferation Kit I, Roche) with a
healthy donors. Cartilage samples from healthy donors NanoQuant microplate reader (Tecan Trading AG) at
were obtained after knee or hip fracture with no history 570 nm.
of joint disease. All samples (healthy and OA) were
analyzed by histological staining and classified Adipogenic differentiation
following the American College of Rheumatology
(ACR) recommendations and clinical classification Adipogenic differentiation was performed as previously
criteria for OA. The chondrocyte cell line T/C-28a2 was described [2]. Cells were incubated with adipogenic
kindly donated by Dr. Goldring (Hospital for Special medium (hMSC Adipogenic Differentiation BullekitTM,
Surgery; New York, USA). Primary chondrocytes and Lonza) for 21 days, with the addition of oleuropein or
T/C-28a2 cells were cultured in Dulbecco’s Modified OE. Adipogenic differentiation was evaluated by the
Eagle’s medium (DMEM) supplemented with 100 RNA expression of PPARγ and by oil red O staining.
U/mL penicillin, 100 µg/mL streptomycin and 10%
foetal bovine serum (FBS). Human mesenchymal stem Osteogenic differentiation
cells (hMSCs) were obtained with signed informed
consent from 4 bone marrow donors (Hospital For osteogenic differentiation, cells were differentiated
Universitario Reina Sofía; Córdoba, Spain) and from for 21 days with osteogenic medium (StemPro®
subcutaneous inguinal fat from 2 healthy individuals Osteogenesis Differentiation Kit, Gibco, Thermo Fisher
(Hospital Universitario Ramón y Cajal; Madrid, Spain). Scientific), with the addition of oleuropein or OE.
hMSCs were cultured in MesenPRO RSTM Medium Osteogenesis was evaluated by the RNA expression of
supplemented with 100 U/ml penicillin and 100 µg/ml OSTCN and by alizarin red S staining.
streptomycin. Synoviocytes (cultured in RPMI 10%FBS
supplemented with 1% insulin-transferrin-selenium, 100 Chondrogenic differentiation
U/mL penicillin and 100 µg/mL streptomycin) and bone
cells (cultured in DMEM supplemented with 100 U/mL Chondrogenesis was performed in OACs and hMSCs as
penicillin, 100 µg/mL streptomycin and 20% cell micromasses (3D) or as a monolayer culture. For the
inactivated FBS) from OA patients were donated by 3D culture, 500000 cells were seeded in non-adherent
Raquel Largo (IIS-Fundación Jiménez Díaz; Madrid, conic tubes and centrifuged at 500 xg for 10 min, resulting
Spain), after signed informed consent. hMSCs, primary in a pellet formation, and cultured in chondrogenic
chondrocytes, T/C-28a2 cells, BC and SC at 70-80% medium (CM; StemPro® Chondrogenesis Differentiation
confluence were treated with oleuropein (Extrasynthese Kit, Gibco, Thermo Fisher Scientific) supplemented with
#0204; Lyon, France) or an olive extract (OE) oleuropein or OE for 30 days. For the monolayer culture,
www.aging-us.com 15893 AGINGcells were differentiated in CM for 7/14 days with conjugated anti-human CD166 (Immunostep,
oleuropein/OE addition. Chondrogenesis was evaluated 1399990314), for 30 min at 4°C, as previously reported
by proteoglycans detection (toluidine blue staining), [2] (Supplementary Table 1). Intracellular Cx43 protein
Col2A1 immunohistochemistry and by ACAN mRNA was detected in paraformaldehyde-fixed cells after
expression. permeabilization with methanol for 30 min at 4ºC.
Finally, cells were incubated with APC-conjugated anti-
Scrape loading/dye transfer (SL/DT) assay human Cx43 antibody (R&D Systems, FAB7737A) for
30 min at 4°C.
Confluent cells were treated with oleuropein or OE for
2 h. Next, a 0.4% (w/v) solution of lucifer yellow (LY) Flow cytometry analysis
(Cell Projects Ltd©) was loaded and two distant scrapes
were made across the culture plate. In order to evaluate 20.000 events were collected on a FACSCaliburTM
GJIC, a score was calculated as the ratio of non-damage (Becton Dickinson) flow cytometer with the CellQuestTM
positive cells for LY to the damaged cells [2, 10]. Pro software. Cell debris was discriminated by the
forward scatter (FSC) and side scatter (SSC) properties of
GJ connectivity by flow cytometry the cells. Data were analyzed with FCS Express 6 Flow
software (De Novo Software). The level of positive
Equal numbers of cells were incubated for 1 h at 37 ºC staining was expressed as the median fluorescence
with either 1 µM of the membrane dye DiI (Invitrogen, intensity (MFI), with unlabelled cells as negative controls.
Thermo Fisher Scientific) or 1 µM calcein-AM Gates were placed based on single-labelled controls and
(Invitrogen, Thermo Fisher Scientific), a cell-permeable by establishing 0.1% as the cut-off point.
dye that is retained in the cytosol and can be transferred
by GJs. Then, cells were washed with PBS and co- Senescence-associated β-galactosidase activity
cultured for 2 h in a 1:4 ratio (calcein-donors:DiI-
recipient cells). The percentage of DiI+Calc+ cells was Flow cytometry analysis of SA-βGal activity with the
analysed by flow cytometry. fluorogenic β-galactosidase substrate fluorescein di-β-
D-galactopyranoside (FDG; Invitrogen, Thermo Fisher
Western blot Scientific) was performed as previously described [2].
Briefly, cells were harvested and incubated with pre-
Protein analysis was performed as previously described warmed 2 mM FDG for 3 min at 37 ºC and fluorescein
[2, 10]. The following primary antibodies were used: α- positivity was analyzed on a BD FACSCalibur TM
tubulin (Sigma-Aldrich, T9026), Cx43 (Sigma-Aldrich, (Becton Dickinson) flow cytometer. SA-βGal activity
C6219), Twist-1 (Santa Cruz Biotechnology, sc-81417), was also measured with the Senescence Cells
CD166 (Santa Cruz Biotechnology, sc-74558), p16INK4A Histochemical Staining Kit (Sigma-Aldrich) according
(Abcam, ab108349), p21 (Santa Cruz Biotechnology, to the manufacturer’s protocol and analyzed after 16 h
sc-6246), p53 (Santa Cruz Biotechnology, sc-126), NF- of staining.
κB (Santa Cruz Biotechnology, sc-8008), lamin A
(Santa Cruz Biotechnology, sc-20680), IL-6 (Sigma- Immunofluorescence and immunohistochemistry
Aldrich, SAB1403971) and COX-2 (Santa Cruz
Biotechnology, sc-166475). Immunofluorescence and immunohistochemistry assays
were performed as previously described [2, 10]. The
Enzyme-linked immunosorbent assay (ELISA) following primary antibodies were used: anti-Cx43
(Sigma-Aldrich, C6129), anti-collagen II (Invitrogen,
Supernatants were collected after a 72 h-treatment with Thermo Fisher Scientific, MA5-12789), anti-Ki-67
oleuropein 10 µM, and diluted in a 1:8 ratio prior to the (BD, 550609), anti-Twist-1 (sc-81417) and anti-NF-κB
measurement. Samples were incubated for 2 h at room (sc-8008) from Santa Cruz Biotechnology. Goat anti-
temperature, and IL-6 was detected using the rabbit FITC-conjugated (F-2765, Invitrogen, Thermo
quantitative sandwich human IL-6 immunoassay (hIL- Fisher Scientific) and goat anti-mouse Alexa 594-
6-EIA-20, MagTag GmbH). Samples were analyzed at conjugated (A-11032, Invitrogen, Thermo Fisher
450 nm in a TECAN plate reader. Scientific) secondary antibodies were used.
Antigen expression analysis by flow cytometry Quantitative PCR
Paraformaldehyde-fixed cells were incubated with TRIzolTM reagent (Invitrogen, Thermo Fisher Scientific)
phycoerythrin (PE)-conjugated anti-human CD105 was used to isolate total RNA, according to the
(Immunostep, 105PE-100T) or allophycocyanin (APC)- manufacturer's instructions. 1 µg of total RNA per
www.aging-us.com 15894 AGINGreaction was used to synthesize cDNA with the transition; FDG: fluorescein-di-D-galactopyranoside; GJs:
SuperScript® VILO™ cDNA Synthesis Kit (Invitrogen, gap junctions; GJIC: gap junction intercellular
Thermo Fisher Scientific). Quantitative PCR was communication; hMSCs: human mesenchymal stem cells;
performed as previously described [2, 10]. Primers are IL-1β: interleukin 1 beta; IL-6: interleukin 6; LY: Lucifer
listed in Supplementary Table 2. yellow; MMP-3: matrix metalloproteinase 3; NF-κB:
nuclear factor kappa-light-chain-enhancer of activated B
Cell transfection cells; OA: osteoarthritis; OACs: osteoarthritic
chondrocytes; OE: olive extract; OM: osteogenic
Cx43 was overexpressed, as previously described [2], in medium; Oleu: Oleuropein; OSTCN: osteocalcin; p16:
the T/C-28a2 chondrocyte cell after transfection with a cyclin-dependent kinase inhibitor 2A; p53: cellular
pIRESpuro2 plasmid construct (Clontech) containing the tumour antigen p53; PPARγ: peroxisome proliferator-
human Cx43 sequence, kindly provided by Arantxa activated receptor gamma; RT-qPCR: real-time
Tabernero (INCL, University of Salamanca, Spain). quantitative polymerase chain reaction; SA-βGal:
Electroporation was performed with the Amaxa® Cell senescence-associated β-galactosidase activity; SASP:
Line Nucleofector® Kit V (Lonza) in a NucleofectorTM senescence-associated secretory phenotype; SC: synovial
2b device (Lonza) following the manufacturer’s cells; TNFα: tumour necrosis factor alpha; Twist-1: twist-
instructions. related protein 1.
Luciferase reporter assay AUTHOR CONTRIBUTIONS
A DNA construct containing the upstream 300 bp- M.V.-E. designed and performed the experiments,
regulatory sequence of the human Cx43 promoter (– analyzed the data and prepared the figures. P.C.-F. and
108, +279, relative to the human Cx43 transcription A.S-T. performed experiments and analyzed data. A.V.-
start site) in a pGL3-Basic vector was kindly donated by V. assisted with cell culture and chondrocyte isolation
Dr. Mustapha Kandouz (Wayne State University, USA). from patients’ cartilage. C.L.P., M.K., A.C.-D. and
The T/C-28a2 chondrocyte cell line was transfected A.C.-C. provided drugs and hMSCs from healthy
with the Amaxa® Cell Line Nucleofector® Kit V in a donors. V.M. analyzed data. E.F. and J.R.C. contributed
NucleofectorTM 2b device. After 24 h, cells were treated clinical and technical advice. J.R.C. also provided
with 10 µM oleuropein and/or 5 µg/ml of oligomycin cartilage tissue from patients and healthy donors. A.B.
(Sigma-Aldrich) for 1 h. For the luminescence analysis, designed, performed and helped with various aspects of
the Firefly Luciferase Assay Kit from Biotium was used the flow cytometry experiments and analysis. M.D.M.
according to the manufacturer’s instructions, and conceived, directed and supervised the study. M.V.-E.
normalized to the total protein content. and M.D.M. wrote the manuscript with input from all
co-authors. All authors reviewed the manuscript.
Statistical analysis
ACKNOWLEDGMENTS
Statistical analysis was performed with GraphPad Prism
software (version 7.0a). Data of the biological and/or We thank members of the CellCOM group for helpful
technical replicates are represented as mean and SD. technical suggestions, María Dolores Álvarez Alvariño
Two-tailed Unpaired Student’s t test was used to and Jesús Loureiro for generously collecting tissue
estimate statistically significant differences between samples in the operating room after surgery. Ángel
two groups. The estimation of the difference of the Concha and the Biobank of A Coruña, María Vázquez
means among groups was compared by one-way and Beatriz Lema for tissue processing and micromass
ANOVA with Bonferroni correction. P values were sectioning. Moisés Blanco for helpful advice for the
provided, with PMineral Metabolism (FEIOMM), grant PRECIPITA- cancer: bridging the gap to the clinic. Oncogene. 2019;
2015-000139 from the FECYT-Ministry of Economy 38:4429–51.
and Competitiveness (to M.D.M.), grant PI16/00035 https://doi.org/10.1038/s41388-019-0741-6
and PI19/00145 from the Health Institute ‘Carlos III’ PMID:30814684
(ISCIII, Spain), the European Regional Development
8. Gago-Fuentes R, Fernández-Puente P, Megias D,
Fund, ‘A way of making Europe’ from the European
Carpintero-Fernández P, Mateos J, Acea B, Fonseca
Union (to M.D.M.) and a grant from Xunta de Galicia
E, Blanco FJ, Mayan MD. Proteomic analysis of
IN607B 2017/21 (to M.D.M.). M.V.-E. was funded with
connexin 43 reveals novel interactors related
a predoctoral (ED481A-2015/188) and a postdoctoral
to osteoarthritis. Mol Cell Proteomics. 2015;
fellowship (IN606B-2019/004) from Xunta de Galicia.
14:1831–45.
P.C.-F. was funded with a postdoctoral fellowship
https://doi.org/10.1074/mcp.M115.050211
(IN606B-2017/014) from Xunta de Galicia.
PMID:25903580
REFERENCES 9. Mayan MD, Carpintero-Fernandez P, Gago-Fuentes R,
Martinez-de-Ilarduya O, Wang HZ, Valiunas V, Brink P,
1. Jeon OH, Kim C, Laberge RM, Demaria M, Rathod S, Blanco FJ. Human articular chondrocytes express
Vasserot AP, Chung JW, Kim DH, Poon Y, David N, multiple gap junction proteins: differential expression
Baker DJ, van Deursen JM, Campisi J, Elisseeff JH. Local of connexins in normal and osteoarthritic cartilage. Am
clearance of senescent cells attenuates the J Pathol. 2013; 182:1337–46.
development of post-traumatic osteoarthritis and https://doi.org/10.1016/j.ajpath.2012.12.018
creates a pro-regenerative environment. Nat Med. PMID:23416160
2017; 23:775–81. 10. Mayan MD, Gago-Fuentes R, Carpintero-Fernandez P,
https://doi.org/10.1038/nm.4324 PMID:28436958 Fernandez-Puente P, Filgueira-Fernandez P, Goyanes
2. Varela-Eirín M, Varela-Vázquez A, Guitián-Caamaño A, N, Valiunas V, Brink PR, Goldberg GS, Blanco FJ.
Paíno CL, Mato V, Largo R, Aasen T, Tabernero A, Articular chondrocyte network mediated by gap
Fonseca E, Kandouz M, Caeiro JR, Blanco A, Mayán MD. junctions: role in metabolic cartilage homeostasis. Ann
Targeting of chondrocyte plasticity via connexin43 Rheum Dis. 2015; 74:275–84.
modulation attenuates cellular senescence and fosters https://doi.org/10.1136/annrheumdis-2013-204244
a pro-regenerative environment in osteoarthritis. Cell PMID:24225059
Death Dis. 2018; 9:1166. 11. Marino AA, Waddell DD, Kolomytkin OV, Meek WD,
https://doi.org/10.1038/s41419-018-1225-2 Wolf R, Sadasivan KK, Albright JA. Increased
PMID:30518918 intercellular communication through gap junctions
3. Alsalameh S, Amin R, Gemba T, Lotz M. Identification may contribute to progression of osteoarthritis. Clin
of mesenchymal progenitor cells in normal and Orthop Relat Res. 2004; 422:224–32.
osteoarthritic human articular cartilage. Arthritis https://doi.org/10.1097/01.blo.0000129346.29945.3b
Rheum. 2004; 50:1522–32. PMID:15187861
https://doi.org/10.1002/art.20269 PMID:15146422 12. Gago-Fuentes R, Bechberger JF, Varela-Eirin M, Varela-
4. Grogan SP, Miyaki S, Asahara H, D’Lima DD, Lotz MK. Vazquez A, Acea B, Fonseca E, Naus CC, Mayan MD.
Mesenchymal progenitor cell markers in human The c-terminal domain of connexin43 modulates
articular cartilage: normal distribution and changes in cartilage structure via chondrocyte phenotypic
osteoarthritis. Arthritis Res Ther. 2009; 11:R85. changes. Oncotarget. 2016; 7:73055–67.
https://doi.org/10.1186/ar2719 PMID:19500336 https://doi.org/10.18632/oncotarget.12197
PMID:27682878
5. Goldring MB, Marcu KB. Cartilage homeostasis in
health and rheumatic diseases. Arthritis Res Ther. 13. Plotkin LI, Bellido T. Beyond gap junctions: Connexin43
2009; 11:224. and bone cell signaling. Bone. 2013; 52:157–66.
https://doi.org/10.1186/ar2592 PMID:19519926 https://doi.org/10.1016/j.bone.2012.09.030
PMID:23041511
6. Montgomery J, Ghatnekar GS, Grek CL, Moyer KE,
Gourdie RG. Connexin 43-based therapeutics for 14. Tsuchida S, Arai Y, Kishida T, Takahashi KA, Honjo K,
dermal wound healing. Int J Mol Sci. 2018; 19:1778. Terauchi R, Inoue H, Oda R, Mazda O, Kubo T. Silencing
https://doi.org/10.3390/ijms19061778 the expression of connexin 43 decreases inflammation
PMID:29914066 and joint destruction in experimental arthritis. J Orthop
Res. 2013; 31:525–30.
7. Aasen T, Leithe E, Graham SV, Kameritsch P, Mayán https://doi.org/10.1002/jor.22263
MD, Mesnil M, Pogoda K, Tabernero A. Connexins in PMID:23165424
www.aging-us.com 15896 AGING15. Gupta A, Niger C, Buo AM, Eidelman ER, Chen RJ, 24. Peilin W, Songsong T, Chengyu Z, Zhi C, Chunhui M,
Stains JP. Connexin43 enhances the expression of Yinxian Y, Lei Z, Min M, Zongyi W, Mengkai Y, Jing X,
osteoarthritis-associated genes in synovial fibroblasts Tao Z, Zhuoying W, et al. Directed elimination of
in culture. BMC Musculoskelet Disord. 2014; 15:425. senescent cells attenuates development of
https://doi.org/10.1186/1471-2474-15-425 osteoarthritis by inhibition of c-IAP and XIAP. Biochim
PMID:25496568 Biophys Acta Mol Basis Dis. 2019; 1865:2618–32.
16. Gurtner GC, Werner S, Barrandon Y, Longaker MT. https://doi.org/10.1016/j.bbadis.2019.05.017
Wound repair and regeneration. Nature. 2008; PMID:31251987
453:314–21. 25. Nogueira-Recalde U, Lorenzo-Gómez I, Blanco FJ, Loza
https://doi.org/10.1038/nature07039 PMID:18480812 MI, Grassi D, Shirinsky V, Shirinsky I, Lotz M, Robbins
PD, Domínguez E, Caramés B. Fibrates as drugs with
17. Loeser RF, Collins JA, Diekman BO. Ageing and the
pathogenesis of osteoarthritis. Nat Rev Rheumatol. senolytic and autophagic activity for osteoarthritis
therapy. EBioMedicine. 2019; 45:588–605.
2016; 12:412–20.
https://doi.org/10.1038/nrrheum.2016.65 https://doi.org/10.1016/j.ebiom.2019.06.049
PMID:27192932 PMID:31285188
26. Yang H, Chen C, Chen H, Duan X, Li J, Zhou Y, Zeng W,
18. Gao SG, Zeng C, Li LJ, Luo W, Zhang FJ, Tian J, Cheng C,
Yang L. Navitoclax (ABT263) reduces inflammation and
Tu M, Xiong YL, Jiang W, Xu M, Lei GH. Correlation
promotes chondrogenic phenotype by clearing
between senescence-associated beta-galactosidase
senescent osteoarthritic chondrocytes in
expression in articular cartilage and disease severity of
osteoarthritis. Aging (Albany NY). 2020; 12:12750–70.
patients with knee osteoarthritis. Int J Rheum Dis.
https://doi.org/10.18632/aging.103177
2016; 19:226–32.
PMID:32611834
https://doi.org/10.1111/1756-185X.12096
PMID:26112901 27. Zhu Y, Doornebal EJ, Pirtskhalava T, Giorgadze N,
Wentworth M, Fuhrmann-Stroissnigg H, Niedernhofer
19. Philipot D, Guérit D, Platano D, Chuchana P, Olivotto E,
LJ, Robbins PD, Tchkonia T, Kirkland JL. New agents
Espinoza F, Dorandeu A, Pers YM, Piette J, Borzi RM,
that target senescent cells: the flavone, fisetin, and the
Jorgensen C, Noel D, Brondello JM. p16INK4a and its
BCL-XL inhibitors, A1331852 and A1155463. Aging
regulator miR-24 link senescence and chondrocyte
(Albany NY). 2017; 9:955–63.
terminal differentiation-associated matrix remodeling
https://doi.org/10.18632/aging.101202
in osteoarthritis. Arthritis Res Ther. 2014; 16:R58.
PMID:28273655
https://doi.org/10.1186/ar4494 PMID:24572376
28. Zheng W, Feng Z, You S, Zhang H, Tao Z, Wang Q, Chen
20. Price JS, Waters JG, Darrah C, Pennington C, Edwards H, Wu Y. Fisetin inhibits IL-1β-induced inflammatory
DR, Donell ST, Clark IM. The role of chondrocyte response in human osteoarthritis chondrocytes
senescence in osteoarthritis. Aging Cell. 2002; 1:57–65. through activating SIRT1 and attenuates the
https://doi.org/10.1046/j.1474-9728.2002.00008.x progression of osteoarthritis in mice. Int
PMID:12882354 Immunopharmacol. 2017; 45:135–47.
21. Loeser RF. Aging and osteoarthritis: the role of https://doi.org/10.1016/j.intimp.2017.02.009
chondrocyte senescence and aging changes in the PMID:28213268
cartilage matrix. Osteoarthritis Cartilage. 2009; 29. Cao X, Luo P, Huang J, Liang C, He J, Wang Z, Shan D,
17:971–79. Peng C, Wu S. Intraarticular senescent chondrocytes
https://doi.org/10.1016/j.joca.2009.03.002 impair the cartilage regeneration capacity of
PMID:19303469 mesenchymal stem cells. Stem Cell Res Ther. 2019;
22. Collins JA, Diekman BO, Loeser RF. Targeting aging for 10:86.
disease modification in osteoarthritis. Curr Opin https://doi.org/10.1186/s13287-019-1193-1
Rheumatol. 2018; 30:101–07. PMID:30867061
https://doi.org/10.1097/BOR.0000000000000456 30. Kang D, Shin J, Cho Y, Kim HS, Gu YR, Kim H,
PMID:28957964 You KT, Chang MJ, Chang CB, Kang SB, Kim JS,
23. Tchkonia T, Zhu Y, van Deursen J, Campisi J, Kirkland JL. Kim VN, Kim JH. Stress-activated miR-204 governs
Cellular senescence and the senescent secretory senescent phenotypes of chondrocytes to promote
phenotype: therapeutic opportunities. J Clin Invest. osteoarthritis development. Sci Transl Med. 2019;
2013; 123:966–72. 11:eaar6659.
https://doi.org/10.1172/JCI64098 https://doi.org/10.1126/scitranslmed.aar6659
PMID:23454759 PMID:30944169
www.aging-us.com 15897 AGING31. Szychlinska MA, Stoddart MJ, D’Amora U, Ambrosio L, cells from repair tissue during the later stages of
Alini M, Musumeci G. Mesenchymal stem cell-based human osteoarthritis. Cell Stem Cell. 2009; 4:324–35.
cartilage regeneration approach and cell senescence: https://doi.org/10.1016/j.stem.2009.01.015
can we manipulate cell aging and function? Tissue Eng PMID:19341622
Part B Rev. 2017; 23:529–39. 40. Kim AC, Spector M. Distribution of chondrocytes
https://doi.org/10.1089/ten.TEB.2017.0083
containing alpha-smooth muscle actin in human
PMID:28514935 articular cartilage. J Orthop Res. 2000; 18:749–55.
32. Xu M, Bradley EW, Weivoda MM, Hwang SM, https://doi.org/10.1002/jor.1100180511
Pirtskhalava T, Decklever T, Curran GL, Ogrodnik M, PMID:11117296
Jurk D, Johnson KO, Lowe V, Tchkonia T, Westendorf JJ, 41. Dowthwaite GP, Bishop JC, Redman SN, Khan IM,
Kirkland JL. Transplanted senescent cells induce an
Rooney P, Evans DJ, Haughton L, Bayram Z, Boyer S,
osteoarthritis-like condition in mice. J Gerontol A Biol Thomson B, Wolfe MS, Archer CW. The surface of
Sci Med Sci. 2017; 72:780–85.
articular cartilage contains a progenitor cell
https://doi.org/10.1093/gerona/glw154 population. J Cell Sci. 2004; 117:889–97.
PMID:27516624 https://doi.org/10.1242/jcs.00912 PMID:14762107
33. Laird DW. Syndromic and non-syndromic disease-
42. Fickert S, Fiedler J, Brenner RE. Identification of
linked Cx43 mutations. FEBS Lett. 2014; 588:1339–48. subpopulations with characteristics of mesenchymal
https://doi.org/10.1016/j.febslet.2013.12.022
progenitor cells from human osteoarthritic cartilage
PMID:24434540
using triple staining for cell surface markers. Arthritis
34. Yang Y, Ren J, Sun Y, Xue Y, Zhang Z, Gong A, Wang B, Res Ther. 2004; 6:R422–32.
Zhong Z, Cui Z, Xi Z, Yang GY, Sun Q, Bian L. A https://doi.org/10.1186/ar1210 PMID:15380042
connexin43/YAP axis regulates astroglial-mesenchymal
43. Jiang Y, Cai Y, Zhang W, Yin Z, Hu C, Tong T, Lu P, Zhang
transition in hemoglobin induced astrocyte activation.
S, Neculai D, Tuan RS, Ouyang HW. Human cartilage-
Cell Death Differ. 2018; 25:1870–84.
derived progenitor cells from committed chondrocytes
https://doi.org/10.1038/s41418-018-0137-0
for efficient cartilage repair and regeneration. Stem
PMID:29880858
Cells Transl Med. 2016; 5:733–44.
35. Varela-Eirin M, Loureiro J, Fonseca E, Corrochano S, https://doi.org/10.5966/sctm.2015-0192
Caeiro JR, Collado M, Mayan MD. Cartilage PMID:27130221
regeneration and ageing: targeting cellular plasticity in
44. Li L, Newton PT, Bouderlique T, Sejnohova M, Zikmund
osteoarthritis. Ageing Res Rev. 2018; 42:56–71.
T, Kozhemyakina E, Xie M, Krivanek J, Kaiser J, Qian H,
https://doi.org/10.1016/j.arr.2017.12.006
Dyachuk V, Lassar AB, Warman ML, et al. Superficial
PMID:29258883
cells are self-renewing chondrocyte progenitors, which
36. Casagrande D, Stains JP, Murthi AM. Identification of form the articular cartilage in juvenile mice. FASEB J.
shoulder osteoarthritis biomarkers: comparison 2017; 31:1067–84.
between shoulders with and without osteoarthritis. J https://doi.org/10.1096/fj.201600918R
Shoulder Elbow Surg. 2015; 24:382–90. PMID:27965322
https://doi.org/10.1016/j.jse.2014.11.039
45. Mori R, Power KT, Wang CM, Martin P, Becker DL.
PMID:25595362
Acute downregulation of connexin43 at wound sites
37. Tonon R, D’Andrea P. The functional expression of leads to a reduced inflammatory response, enhanced
connexin 43 in articular chondrocytes is increased by keratinocyte proliferation and wound fibroblast
interleukin 1beta: evidence for a Ca2+-dependent migration. J Cell Sci. 2006; 119:5193–203.
mechanism. Biorheology. 2002; 39:153–60. https://doi.org/10.1242/jcs.03320 PMID:17158921
PMID:12082278
46. Pollok S, Pfeiffer AC, Lobmann R, Wright CS, Moll I,
38. Aigner T, Zien A, Gehrsitz A, Gebhard PM, McKenna L. Martin PE, Brandner JM. Connexin 43 mimetic peptide
Anabolic and catabolic gene expression pattern Gap27 reveals potential differences in the role of Cx43
analysis in normal versus osteoarthritic cartilage using in wound repair between diabetic and non-diabetic
complementary DNA-array technology. Arthritis cells. J Cell Mol Med. 2011; 15:861–73.
Rheum. 2001; 44:2777–89. https://doi.org/10.1111/j.1582-4934.2010.01057.x
https://doi.org/10.1002/1529-0131(200112)44:12 PMID:20345849
3.0.co;2-h PMID:11762938 47. Ongstad EL, O’Quinn MP, Ghatnekar GS, Yost MJ,
39. Koelling S, Kruegel J, Irmer M, Path JR, Sadowski B, Gourdie RG. A Connexin43 mimetic peptide promotes
Miro X, Miosge N. Migratory chondrogenic progenitor regenerative healing and improves mechanical
www.aging-us.com 15898 AGINGYou can also read
