PLOD2, a key factor for MRL MSC metabolism and chondroprotective properties

doi:10.21203/rs.3.rs-2733110/v1

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PLOD2, a key factor for MRL MSC metabolism and chondroprotective properties

https://doi.org/10.21203/rs.3.rs-2733110/v1

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published 07 Mar, 2024

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Background: Initially discovered for its ability to regenerate ear holes, the MRL mouse has been the subject of multiple research studies aimed at evaluating its ability to regenerate other body tissues and at deciphering the mechanisms underlying it. These enhanced abilities to regenerate, retained in the adult, protect the MRL mouse from degenerative diseases such as osteoarthritis (OA). Here, we hypothesized that MSC derived from the regenerative MRL mouse could be involved in their regenerative potential through the release of pro-regenerative mediators.

Method: To address this hypothesis, we compared the transcriptome of MRL and BL6 MSC and identified several candidate molecules expressed at significantly higher levels by MRL MSC than by BL6 MSC. We selected one candidate and performed functional in vitro assays to evaluate its role on MRL MSC properties including metabolic profile, migration, and chondroprotective effects. Using an experimental model for osteoarthritis (OA) induced by collagenase (CiOA), we assessed its contribution to MRL MSC protection from OA.

Results: Among the candidate molecules highly expressed by MRL MSC, we focused our attention on procollagen-lysine,2-oxoglutarate 5-dioxygenase 2 (PLOD2), coding for the lysyl hydrolase LH2 in charge of post-translational modifications of collagen for its stability and stiffness. PLOD2 is induced by hypoxia-inducible factor 1-alpha (HIF-1a) involved in the regeneration process of adult MRL mice. Plod2 silencing induced a decrease in the glycolytic function of MRL MSC, resulting in the alteration of their migratory and chondroprotective abilities in vitro. In vivo, we showed that plod2 silencing in MRL MSC significantly impaired their capacity to protect mouse from developing OA.

Conclusion: Our results demonstrate that the chondroprotective and therapeutic properties of MRL MSC in the CiOA experimental model are in part mediated by PLOD2.

MRL mouse

regeneration

mesenchymal stem cells

PLOD2

metabolism

chondroprotection

Regeneration ability is a property that varies widely during development and among species. Indeed, while able to regenerate at early embryonic stages, adult mammals will trigger a tissue repair mechanism resulting in scar formation after tissue injury and limiting the structural and functional recovery [1, 2]. In contrast, some species, such as salamander [3], zebrafish [4] and hydra [5, 6] maintain their regenerative properties during their all-life [7–9]. Rare exceptions, such as the super healer Murphy Roths Large (MRL) mouse, exist among mammals. Indeed, the adult MRL mouse is a competent model for tissue regeneration, suggesting that crucial regenerative mechanisms are conserved in this mammalian model. MRL mouse possesses the extraordinary potential to regenerate multiple musculoskeletal tissues such as the outer ear, articular cartilage, and digits without scarring [10–15]. Among the conserved mechanisms underlying regeneration, the emphasis on aerobic glycolytic energy metabolism has been reported to be essential in several regenerative species [16–18].

MRL mice are protected from the development of joint diseases such as osteoarthritis (OA) [11, 19]. Identifying the mechanisms underlying articular cartilage regeneration and protection from OA would allow the development of novel therapies for OA patients. Indeed, OA is a complex disease characterized, in part, by the degradation of articular cartilage and for which no curative treatment exists to date. One therapeutic option studied is the intra-articular administration of mesenchymal stem/stromal cells (MSC). The trophic activities of MSC introduced exogenously have been shown to protect from cartilage degradation and OA development in experimental model of OA [20–22]. Indeed, such as resident, exogenous MSC participate in joint homeostasis through a paracrine action that contributes to the repair of damaged tissue, the restoration of tissue metabolism and the prevention of inflammation [22, 23].

In the context of digit tip regeneration in mice, mesenchymal cells from one type of tissue were shown to participate to the regeneration of other mesenchymal tissues [24]. The authors showed that the regenerative environment primes the cells from injured tissues to acquire a blastema mesenchymal transcriptional state enabling them to regenerate other mesenchymal tissues such as the dermis. Therefore, it can be speculated that the identification of regenerative environmental factors could stimulate the regenerative potential of MSC in adult mammals. The use of adult MSC in experimental models of degenerative diseases such as OA have shown promising results [20, 25, 26]. However, while MSC prevent cartilage degradation and OA development when administrated locally their capacity to regenerate damaged OA cartilage has never been proven.

The cartilage regenerative potential of MRL mice and their subsequent resistance to experimental osteoarticular defects has increased the interest in MSC derived from MRL mice (MRL MSC). Therefore, we hypothesized that MRL MSC regenerative and protective properties might be associated with their intrinsic properties, particularly their aerobic glycolytic energy metabolism controlled via soluble factors. To address that hypothesis and identify soluble factors presumably at the origin of MRL mouse resistance to OA, we performed a comparative study of MRL MSC and BL6 MSC secretome. Taking into account the glycolytic metabolic profile of the MRL mouse, we investigated the role of one of the genes overexpressed by MRL MSC: Plod2, in their regenerative capacities.

MSC isolation and expansion

Mesenchymal Stem/Stromal Cells (MSC) from MRL/Mpj (MRL MSC) and C57BL/6 (BL6 -MSC) mice bone marrow were isolated and expanded as previously described [27].

Murine chondrocyte culture and co-culture

Murine articular chondrocytes were isolated from the knees and femoral head of 3-day-old C57BL/6 mice as described previously [28, 29]. Briefly, chondrocytes (25 000 cells/cm²) were plated in 12-well culture plates (TPP Techno Plastic Products, Switzerland) with 1 mL of proliferative medium for five days. Then, chondrocytes were treated with 1ng/mL Il-1b (R&D Systems) for 24h (day 0) to generate the so-called "OA-like" chondrocytes. For co-culture experiments, 2 x10⁵of naïve or modified MSC were seeded in 12-well culture inserts with 1 mL of proliferative media and co-cultured with OA-like chondrocyte for 24h (day 1). 48 hours later, chondrocytes were recovered (day 3) and processed for RT-qPCR.

MSC transfection with siRNA for PLOD2 silencing

MRL MSC were transfected overnight at subconfluence (45%) with 20mM of control siRNA (siCTL) or the siRNA against PLOD2 (siPLOD2) (Silencer® pre-designed siRNA, Ambion, Life Technologies™) using Oligofectamine reagent (Life Technologies, Courtaboeuf) according to the supplier's recommendations. Transfected MRL MSC were used for follow-up experiments 24H post-transfection.

MSC transfection with CMV plasmid for PLOD2 overexpression

MSC derived from BL6 mice were transfected overnight at subconfluence (60%) with 7,5 ng of PLOD2 plasmid (CMV PLOD2) (pRP[Exp] -mCherry-CMV>mPLOD2 [NM_001142916.1], Vector Builder) using Lipofectamine reagent (Life Technologies, Courtaboeuf) following to the supplier's recommendations. Transfection level was assessed by using fluorescent microscope (ThermoFisher EVOS™ M5000 Imaging System). Transfected MSC positive for mCherry were used for experiment 24 hours post-transfection.

RT-qPCR

Total RNA was isolated from mMSC or chondrocytes using the RNeasy Mini Kit (Qiagen, Courtaboeuf), and the quantity and purity of the total RNA were determined using a NanoDrop ND-1000 spectrophotometer (NanoDrop ND, Thermo Fisher Scientific). cDNA was synthesized by reverse transcribing 500ng of RNA into cDNA using the SensiFAST™ cDNA Synthesis Kit (Bioline, Meridian Life Science© Company). Quantitative PCR was performed on 6,25ng of cDNA using the SensiFAST™ SYBR® No-ROX kit (Bioline, Meridian Life Science© Company) and a LightCycler^® 480 Detection system (Roche), following manufacturer's recommendations. Specific primers for mouse Plod2, Cspg4, Inhbb, Efemp1, Lama4, Mmp3, Htra1, Nt5e, Lcn2, C1qtnf5, Fam20c and Hif-1α were designed using the Primer3 software and can be provided upon reasonable request. Primers for Col2b, Agn, Mmp13, Adamts5 are the same as previously described [29]. Values were expressed as relative mRNA level of specific gene expression as obtained using the 2^−ΔCt method, using the Rsp9 and Actin expression as housekeeping genes.

Scratch wound healing

Migratory potential of the cells was assessed with scratch wound healing assay. 2.5x10⁵cells were seeded in TC24 plates and maintained at 37 °C with 5% of CO2 in proliferating media. The wound was performed manually once the cells adhered to the plastic and reached 90% confluency. Wound closure was studied using an inverted microscope (EVOS M5000, Thermo Fisher Scientific) and images of the scratch were taken at H0, just after the scratch and at H24 to evaluate the wound closure. The wounded area was measured at H0 and H24 using Image J Software: the open wound area (in percentage) was calculated by comparing H0 and H24 images and normalized to H0.

Seahorse assay

Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using the XF96 analyzer (Seahorse Biosciences, North Billerica, MA, USA). Transfected and non-transfected murine MSCs (20-25,000 cells/well) were plated on 96-well plates the day before the experiment in XF media (non-buffered DMEM medium, containing 25 mM glucose, 2 mM L-glutamine, and 1 mM sodium pyruvate). The analysis was conducted according to the manufacturer's recommended protocol. Three independent readings were taken after each sequential injection. The instrumental background was measured in separate control wells using the same conditions without biologic material. The basal glycolytic rate was calculated after glucose injection. The maximal glycolytic rate was measured after oligomycin injection and glycolytic capacity as the difference of oligomycin-induced ECAR and basal stage. OCR was measured under basal conditions and in response to 1 μM of oligomycin, 1 μM of carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP) (Seahorse XF Cell Energy Phenotype Test Kit from Agilent or Sigma Aldrich), and 1 μM of antimycin A and rotenone (Sigma Aldrich). Basal OCR was calculated as the difference between baseline measurements and antimycin A/rotenone-induced OCR; maximal OCR was measured as the difference between FCCP-induced OCR and antimycin A/rotenone-induced OCR. ECAR/OCR ratio was calculated with the glycolytic rate and basal OCR. After the seahorse experiment, the plated cells were fixed for 20 minutes in 4% PFA and then stained with HOESCHT (1/2000eme) to count cells with High-Content Analysis Cellomics™ instrument (Thermo Fisher Scientific) for normalization.

Collagenase-induced osteoarthritis (CiOA) mouse model and histological analysis

Mice used for this study were housed and cared in accordance with the European directive 2010/63/EU. The CiOA model was generated upon the approval from the Ethical Committee for animal experimentation of the Languedoc-Roussillon and the French Ministry for Higher Education and Research (Approval #5349-2016050918198875 v3). Briefly, 1U type VII collagenase in 5 μL saline was administrated in the intra-articular (IA) space of C57BL/6 mice knee joints (10 weeks old) at day 0 and 2. Groups of 10 mice received MSC (2.5 × 105 cells/5 μL saline) at day 7. At day 42, mice were euthanatized by exposure to CO₂until complete cessation of breathing was observed followed by cervical dislocation, and paws were recovered for fixation in 4% formaldehyde and decalcified in 4% EDTA solution for three weeks before paraffin embedding. Tibias were sectioned frontally as previously described [20, 29, 30] and stained with safranin O fast green. Two persons performed blind quantification of the degradation of cartilage using the modified Pritzker OARSI score as described [20, 29, 30]. Mice corresponding to uninterpretable stained slides were removed from the analysis.

Statistical analysis

All data are presented as the mean ± Standard Error of the Mean (SEM), and all experiments were performed at least three times. The Student's t-test was used to compare two experimental groups, and ANOVA followed by a Friedman test for multi comparison of paired samples was used for the co-culture experiments while ANOVA with Kruskal-Wallis test for multiple comparisons of non-paired samples was used for the CiOA. Graphs show mean ±Standard SEM. P-values < 0.05 (*), P < 0.01 (**) or P < 0.001 (***) were considered statistically signiﬁcant. Analysis and graphical representation were performed using Graph-Pad Prism^TM software (Graphpad)

MRL MSC exhibit a specific secretome as compared to MSC derived from C57BL/6 mice

We recently performed label-free quantitative shotgun proteomics to identify differentially secreted proteins between MRL MSC and BL6 MSC [30]. This published secretome was analyzed denovo on the basis of protein intensities quantified by LC-MS/MS with the aim to identify key factors for MRL MSC metabolism and chondroprotective properties (Fig. 1A). Among the 810 proteins differentially expressed between MRL MSC and BL6 MSC by at least 1.5‐fold, 625 proteins were secreted at higher levels by MRL MSC. We focused our attention on proteins with a higher secretory profile by MRL MSC in particular LAMA4, HTRA1, PLOD2, INHBB, MMP3, CSPG4, NT5E, LCN2, EFEMP1, FAM20C and C1QTNF5 (Fig. 1B). By RT-qPCR we confirmed that these 11 factors were overexpressed at a significantly higher level in MRL MSC as compared to BL6 MSC (Fig. 1C).

Plod2 is required for MRL MSC glycolytic metabolism

MRL mouse use of aerobic glycolysis as their basal metabolic state, characterized by the increased production levels of lactate by cells and in serum, has been proposed [31, 32]. First, we wondered whether MRL MSC exhibit a different metabolism than BL6 MSC. To address that question, we compared the oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR) of the two types of MSC by performing a Seahorse assay (Fig. 2A) We found a significantly lower OCR in MRL MSC (Fig 2B and 2D) while no difference was observed for the ECAR, which can be interpretated as the glycolysis rate (Fig 2C and 2E) . The ratio of ECAR to OCR, was significantly higher in MRL MSC than in MSC BL6 suggesting an increased glycolytic function in MSC MRL(Fig. 2F). We then, wondered which factor highly expressed by MRL MSC could be responsible for their glycolytic status. Among the protein overexpressed by MRL MSC as compared to BL6 MSC, we focused our attention on PLOD2 known for promoting aerobic glycolysis in cancer cells [33]. To investigate the role of PLOD2, we used the small interfering RNA (siRNA) approach to knock down the expression of plod2 in MRL MSC. 48 hours post-transfection of MSC with a siRNA against plod2 (siPLOD2), plod2 expression was reduced by 67% compared with the MSC transfected with the control siRNA (siCTL) (Supplementary Figure 1A). We quantified, in MRL MSC either transfected with the siCTL (MRL) or the siPLOD2 (MRL siPLOD2) the OCR, associated to oxidative phosphorylation, and found that plod2 silencing significantly increased OCR in MRL MSC (Fig. 2G and 2I). ECAR assessment under the same conditions showed that plod2 silencing did not affect MRL MSCsi PLOD2 when compared to MRL MSC transfection with the control (Fig. 2H and 2J). Overall, the knockdown of plod2 in MRL MSC induces a switch from glycolysis to OXPHOS as confirmed by the significantly lower ratio of ECAR to OCR in MRL MSC deficient for plod2 than in MSC transfected with siCTL (Fig. 2K).

Conversely, we asked whether plod2 overexpression in BL6 MSC would further enhance their glycolytic activity. To address that question, BL6 MSC were transfected with either a plasmid encoding plod2 (BL6 + CMV_PLOD2) or an empty vector control (BL6) (Supplementary Figure 1B and 1C) and evaluated the OCR or the ECAR of the cells with Seahorse analyzer. Plod2 overexpression in BL6 MSC did not modify the OCR (Fig. 2M and 2L) while inducing a switch from OXPHOS to glycolysis as confirmed by the significantly higher ECAR (Fig. 2N -O) and ratio of ECAR to OCR in BL6 MSC overexpressing plod2 than in control BL6 MSC (Fig. 2P). Altogether, these results provide evidence for the role of plod2 in the cytosolic glycolytic activity in MSC.

Plod2 is required for MRL MSC migration potential

Since a clear relationship between MSC migration and tissue repair has been established [34], we have recently studied the migration potential of MRL MSC and showed that MRL MSC display a significantly higher migration potential than BL6 MSC [30]. To specifically study the role of plod2 on MRL MSC migratory potential in vitro, we analyzed in a scratch wound assay the non-directional migration of MRL and MRL siPLOD2 MSC by evaluating the area of the wound at 24-hours post-wounding using Image J software (National Institutes of Health, Bethesda, MD, USA) (Fig. 3A). Representative images from scratch wound healing assay, 24 hours post-wounding, indicated an altered resurfacing of the wound for MRL siPLOD2 MSC as compared to MRL MSC (Fig. 3B). The percentage of the open wound area at 24 hours which reflects the migration potential of the cells confirmed that MRL siPLOD2 MSC closed the wound significantly slower than MRL MSC (Fig. 3C). Conversely, plod2 overexpression in BL6 MSC significantly increased the migration potential of BL6 MSC (Fig. 3D) and decreased the open wound area (Fig. 3E). Altogether these results indicate that plod2 expression plays a positive role on the migration potential MSC.

Plod2 is necessary for MRL MSC chondroprotective properties

MSC protect chondrocytes from the loss of their mature and functional phenotype in vitro and in vivo [25, 29, 35, 36]. Recently, we have shown that pycr1, pivotal for MRL MSC glycolysis, contributed to their pro-anabolic function on chondrocytes [37]. We then wondered whether plod2 highly produced by MRL MSC could protect chondrocyte from a loss of anabolic markers in vitro. To that end, we relied on coculture experiments with MSC and IL-1β-induced chondrocytes in which chondrocytes exhibit a loss of their anabolic markers including Col2B and Acan and an increase of their catabolic markers and compared the chondroprotective potential of naïve or genetically modified MRL and BL6 MSC (Fig. 4A). First, we tested the effect of plod2 silencing on MRL MSC chondroprotective effects on the IL-1β-induced chondrocyte model. While co-culture of IL-1β-treated chondrocytes with MRL MSC transfected with a siCtl (mMSC MRL) tend to protect the chondrocytes from a loss of Col2B (Fig. 4B), an anabolic marker MRL MSC silenced for plod2 (mMSC MRL_siPLOD2) did not (Fig. 4C). Moreover, while co-culture of IL-1β-treated chondrocytes with MRL MSC transfected with a siCtl (MRL MSC) tend to protect the chondrocytes from an increase of ADAMTS5, a catabolic marker, MRL MSC silenced for plod2 (mMSC MRL_siPLOD2) did not (Fig. 4C). Conversely, plod2 overexpression in BL6 MSC (mMSC BL6_CMV-PLOD2) significantly protect the chondrocytes from an ADAMTS5 increase (Fig. 4D). Altogether, these data show that plod2 tend to protect MSC from the loss of mature chondrocyte phenotype and the increased expression of catabolic markers which are characteristics of OA.

MRL MSC protection against OA is mediated by plod2

We subsequently investigated the role of plod2 on MRL MSC chondroprotective ability in vivo. For this purpose, we used the collagenase-induced osteoarthritis (CiOA) model, in which mice show signs of cartilage degradation, to test the effect of intra-articular injection of MRL MSC transfected with siCTL (MSC MRL) or siPLOD2 (MSC MRL_siPLOD2). At D42, histological examination revealed a lower osteoarthritis score in collagenase-treated mice injected with MRL MSC than in collagenase-treated mice without MRL MSC, indicating a protective effect of the MRL MSC (Figure 5A and 5B). In contrast, the OA score of mice injected with MRL MSCsilenced for plod2 (MSC MRL_siPLOD2) was significantly higher than that of untreated mice (injected with PBS) or CiOA mice treated with control MRL MSC (Figure 5A and 5B). Those results suggest that plod2 contributes to the protective effect of MSC MRL against OA.

Unlike most mammals, the “super healer” MRL mouse retains its regenerative capacity in adulthood. Indeed, this mouse can regenerate, among other tissues, its muscles, its nervous system, and its cartilage. This particularity could explain why this animal model does not develop specific degenerative pathologies such as osteoarthritis or osteoporosis when induced experimentally. To better understand their resistance to joint degenerative diseases we focused our attention on the secretome of their MSC in comparison with BL6 MSC and demonstrate a specific secretome of MRL MSC with a significantly higher production of PLOD2 compared to BL6 MSC. Plod2 produced by MRL MSC assigns them their glycolytic status as well as their chondroprotective properties.

PLOD2, involved in lysyl hydroxylation of collagen molecule pivotal for the stability of collagen cross-links [38], is regulated by hypoxia-inducible factor (HIF)-1α [39, 40]. While the majority of research investigating the contribution of PLOD2 in tumorigenesis [41, 42], our study provides the first evidence of the role of PLOD2 on MRL MSC metabolism, migration potential and chondroprotective properties. HIF1α plays a crucial role in MSC functions [43] and several if not all mammalian regeneration processes [44]. In MRL mice, after tissue wounding, the biphasic expression profile of HIF1α, characterized by a rapid increase of systemic HIF1α levels peaking between days 10 and 14 and decreasing during the second of the regeneration process, suggested the critical role of HIF1α during the process [44, 45]. This was confirmed in experiments showing that hif-1α silencing in MRL mice inhibited ear hole closure and that the injection of drugs stabilizing HIF1α in a hydrogel both proximal and distal to the injured sites led to an accelerated ear hole closure [44, 46]. Since HIF1α is a regulator of PLOD2 expression [47] [48], we assessed the expression level of hif-1α in MRL MSC silenced for plod2 and found that hif-1α was reduced in MRL MSC transfected with the siRNA against plod2 as compared to MRL MSC transfected with the siCTL (Supplementary Fig. 2A and 2B). Conversely, plod2 overexpression in BL6 MSC causes a metabolic switch from oxidative phosphorylation to glycolysis. Altogether, these results suggest that in MRL MSC PLOD2 regulates hif-1α expression and that the PLOD2- HIF1α axis controls MRL MSC glycolysis and regenerative properties we have described [37] and recent findings in the literature showing the induction of PLOD2 by the HIF1α axis for ECM remodeling [40].

In vitro, in gain and loss of function experiments, we have shown that plod2 expression increases MRL MSC migration. These results are in line with a study showing that the inhibition of hypoxia-induced PLOD2 reduces the migration and the invasion of glioma cell both in vitro and in vivo [49]. This was associated with an elevated expression of E-cadherin and reduced expression of vimentin, N-cadherin, snail and slug in response to PLOD2 suppression. Further experiments should be performed to determine whether the decreased migration potential of MRL MSC in response to plod2 silencing is due a modulation of adhesion molecule expression levels.

The silencing of plod2 in MRL MSC also altered MSC chondroprotective properties on IL-1β-treated chondrocytes. Indeed, we demonstrated that while MRL MSC protects IL-1β-treated chondrocytes from a loss of anabolic marker Col2B, MRL MSC silenced plod2 did not. These results are consistent with our results in the CiOA model where MSC MRL downregulated for plod2 lose their chondroprotective effect. Therefore, we propose that the chondroprotective effect of MRL MSC relies, in part, on plod2 overexpression.

Our results are in contradiction with Bank et al. study suggesting that PLOD2 inhibition and therefore the prevention of the formation of pyridinoline cross-links which stabilize the collagen and, might favor cartilage repair attempts. They argue that by showing cartilage with collagen-containing low levels of hydroxylysine and pyridinoline might be less prone to degradation induced mechanically [50]. Therefore, the positive role of plod2 expression on MRL MSC chondroprotective effect might be due to other properties of plod2 than that to form collagen cross-links. Stegen and colleagues recently showed that collagen synthesis in chondrocytes was metabolically controlled by hif-1a and plod2 post-translational modifications [51]. However, sustained expression of plod2 can lead to bone dysplasia, suggesting its involvement in fibrosis [51]. Interestingly, overexpression of PLOD2 via the TGF-1 pathway in adipose tissue-derived MSC increases the therapeutic potential of MSC in an experimental model of spinal cord injury [52]. In mice with a dominant-negative mutation of the TGF- type II receptor, disorganization of collagen fibers was observed [53]. In view of the discrepancy in these results, it would be interesting to know whether the TGF pathway is also used by the MRL mouse for PLOD2 induction and whether this is beneficial against OA.

We have recently shown that the chondroprotective effect of MRL MSC has been associated with their glycolysis [37]. Indeed, we showed that Pyrroline-5-Carboxylate Reductase 1 (Pycr1) downregulation induced MRL MSC metabolism reprogramming specifically associated with a reduced lactate concentration in the extracellular media of the cells. This OXPHOS metabolic reprogramming of MRL MSC knockdown for Pycr1 induced a loss of MRL MSC chondroprotective functions. It is thus reasonable to propose that PLOD2 is pivotal for MRL MSC chondroprotective functions through the high glycolytic flux produced by MRL MSC.

In conclusion, our findings demonstrate for the first time that the enhanced chondroprotective potential of MRL MSC is attributed, in part, to PLOD2, which controls their higher glycolytic potential compared to BL6 MSC.

CiOA: Collagenase-induced osteoarthritis

MRL: Murphy Roths Large

MSC: Mesenchymal stromal/stem cells

ECAR: extracellular acidification rate

HIF-1a: hypoxia-inducible factor 1-alpha

OA: osteoarthritis

OCR: Oxygen consumption rate

PLOD2: procollagen-lysine,2-oxoglutarate 5-dioxygenase 2

RT-PCR: reverse transcription-polymerase chain reaction

SEM: Standard Error of the Mean

Ethics approval

The CiOA model was generated upon the approval from the Institutional Approval Ethical Committee named “Comité d'éthique en expérimentation animale N°036” for animal experimentation of the Languedoc-Roussillon and the French Ministry for Higher Education and Research (Approval number 5349-2016050918198875 v3). The approved project entitled « Effet thérapeutique des cellules souches mésenchymateuses par ingénierie tissulaire ou thérapie cellulaire dans l’arthrose » was approved in March 2017 for a period of 5 years (period during which we carried out our experiments presented here).

Consent for publication

« Not applicable »

Availability of data and material

All in vivo protocols were approved by the Comité d'éthique en expérimentation animale N°036 of the Languedoc-Roussillon and the French Ministry for Higher Education and Research as part of the « Effet thérapeutique des cellules souches mésenchymateuses par ingénierie tissulaire ou thérapie cellulaire dans l’arthrose » project on the March 24^th, 2017 and valid for 5 years. Approval number : AP AFIS#5349-20 16050918198875 v3

The datasets during and/or analyzed during the current study available from the corresponding author on reasonable request.

Competing interests

The authors declare no competing interests

Funding Sources

This work was supported by Inserm, the University of Montpellier. SB was financed by ANRT and CellVax company.

Declaration of Financial Interests

The authors declare no competing financial interests.

Acknowledgements

We thank the MRI facility for their assistance and Daouda Abba Moussa from Metamontp for his help in the Seahorse experiment. We thank the Réseau d’Histologie Expérimentale de Montpellier (Plateau RHEM) histology facility for tissue processing and the animal facility RAM-Neuro for his help in animal breeding and handling. All the illustrations were created with Biorender.com

Author Contributions

FD and SB designed the whole project and the experiments with input of FA, GT.

SB performed the experiments and analysed the results with the input of GT, A-L.M-B and FA.

FD and SB wrote the manuscript with the input of CJ, MW, FA.

All authors read and approved the final manuscript.

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Supplementary figure 1. siRNA and plasmid transfection control (A) RT-qPCR analysis of Plod2 in MRL MSC transfected with control (siCTL) or anti-PLOD2 siRNA (siPLOD2) 24h and 48h post-transfection (n=1) (B) RT-qPCR analysis of Plod2 in BL6 MSC transfected with plasmid CMV PLOD2-mCherry (BL6+CMV PLOD2). (C) BL6 MSC transfection with CMV PLOD2 was assessed for mCherry expression. Supplementary figure 2. Hif -1a mRNA levels in MRL MSC (A) RT-qPCR analysis of Hif-1a in MRL MSC transfected with control (siCTL) or anti-PLOD2 siRNAs (siPLOD2) 24h post-transfection (n=1) (B) RT-qPCR analysis of Hif-1a in BL6 MSC and MRL MSC Error bars represent mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001, Mann-Whitney unpaired t-test, two-tailed (n=3

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PLOD2, a key factor for MRL MSC metabolism and chondroprotective properties

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