Pro-Inflammatory Cytokine Priming and Purification Method Modulate the Impact of Exosomes Derived from Equine Bone Marrow Mesenchymal Stromal Cells on Equine Articular Chondrocytes.

Osteoarthritis (OA) is a widespread osteoarticular pathology characterized by progressive hyaline cartilage degradation, exposing horses to impaired well-being, premature career termination, alongside substantial financial losses for horse owners. Among the new therapeutic strategies for OA, using mesenchymal stromal cell (MSC)-derived exosomes (MSC-exos) appears to be a promising option for conveying MSC therapeutic potential, yet avoiding the limitations inherent to cell therapy. Here, we first purified and characterized exosomes from MSCs by membrane affinity capture (MAC) and size-exclusion chromatography (SEC). We showed that intact MSC-exos are indeed internalized by equine articular chondrocytes (eACs), and then evaluated their functionality on cartilaginous organoids. Compared to SEC, mRNA and protein expression profiles revealed that MAC-exos induced a greater improvement of eAC-neosynthesized hyaline-like matrix by modulating collagen levels, increasing PCNA, and decreasing Htra1 synthesis. However, because the MAC elution buffer induced unexpected effects on eACs, an ultrafiltration step was included to the isolation protocol. Finally, exosomes from MSCs primed with equine pro-inflammatory cytokines (IL-1ß, TNF-α, or IFN-γ) further improved the eAC hyaline-like phenotype, particularly IL-1ß and TNF-α. Altogether, these findings indicate the importance of the exosome purification method and further demonstrate the potential of pro-inflammatory priming in the enhancement of the therapeutic value of MSC-exos for equine OA treatment.

Early Castration in Horses Does Not Impact Osteoarticular Metabolism.

The castration of stallions is traditionally performed after puberty, at around the age of 2 years old. No studies have focused on the effects of early castration on osteoarticular metabolism. Thus, we aimed to compare early castration (3 days after birth) with traditional castration (18 months of age) in horses. Testosterone and estradiol levels were monitored from birth to 33 months in both groups. We quantified the levels of biomarkers of cartilage and bone anabolism (CPII and N-MID) and catabolism (CTX-I and CTX-II), as well as of osteoarthritis (HA and COMP) and inflammation (IL-6 and PGE2). We observed a lack of parallelism between testosterone and estradiol synthesis after birth and during puberty in both groups. The extra-gonadal synthesis of steroids was observed around the 28-month mark, regardless of the castration age. We found the expression of estrogen receptor (ESR1) in cartilage and bone, whereas androgen receptor (AR) expression appeared to be restricted to bone. Nevertheless, with respect to osteoarticular metabolism, steroid hormone deprivation resulting from early castration had no discernable impact on the levels of biomarkers related to bone and cartilage metabolism, nor on those associated with OA and inflammation. Consequently, our research demonstrated that early castration does not disrupt bone and cartilage homeostasis.

Bone Marrow MSC Secretome Increases Equine Articular Chondrocyte Collagen Accumulation and Their Migratory Capacities.

Equine osteoarthritis (OA) leads to cartilage degradation with impaired animal well-being, premature cessation of sport activity, and financial losses. Mesenchymal stem cell (MSC)-based therapies are promising for cartilage repair, but face limitations inherent to the cell itself. Soluble mediators and extracellular vesicles (EVs) secreted by MSCs are the alternatives to overcome those limitations while preserving MSC restorative properties. The effect of equine bone marrow MSC secretome on equine articular chondrocytes (eACs) was analyzed with indirect co-culture and/or MSC-conditioned media (CM). The expression of healthy cartilage/OA and proliferation markers was evaluated in eACs (monolayers or organoids). In vitro repair experiments with MSC-CM were made to evaluate the proliferation and migration of eACs. The presence of nanosized EVs in MSC-CM was appraised with nanoparticle tracking assay and transmission electron microscopy. Our results demonstrated that the MSC secretome influences eAC phenotype by increasing cartilage functionality markers and cell migration in a greater way than MSCs, which could delay OA final outcomes. This study makes acellular therapy an appealing strategy to improve equine OA treatments. However, the MSC secretome contains a wide variety of soluble mediators and small EVs, such as exosomes, and further investigation must be performed to understand the mechanisms occurring behind these promising effects.

Functionalized Nanogels with Endothelin-1 and Bradykinin Receptor Antagonist Peptides Decrease Inflammatory and Cartilage Degradation Markers of Osteoarthritis in a Horse Organoid Model of Cartilage.

Osteoarthritis (OA) is a degenerative and heterogeneous disease that affects all types of joint structures. Current clinical treatments are only symptomatic and do not manage the degenerative process in animals or humans. One of the new orthobiological treatment strategies being developed to treat OA is the use of drug delivery systems (DDS) to release bioactive molecules over a long period of time directly into the joint to limit inflammation, control pain, and reduce cartilage degradation. Two vasoactive peptides, endothelin-1 and bradykinin, play important roles in OA pathogenesis. In this study, we investigated the effects of two functionalized nanogels as DDS. We assessed the effect of chitosan functionalized with a type A endothelin receptor antagonist (BQ-123-CHI) and/or hyaluronic acid functionalized with a type B1 bradykinin receptor antagonist (R-954-HA). The biocompatibility of these nanogels, alone or in combination, was first validated on equine articular chondrocytes cultured under different oxic conditions. Further, in an OA equine organoid model via induction with interleukin-1 beta (IL-1ß), a combination of BQ-123-CHI and R-954-HA (BR5) triggered the greatest decrease in inflammatory and catabolic markers. In basal and OA conditions, BQ-123-CHI alone or in equimolar combinations with R-954-HA had weak pro-anabolic effects on collagens synthesis. These new nanogels, as part of a composite DDS, show promising attributes for treating OA.

Marine Collagen Hydrolysates Downregulate the Synthesis of Pro-Catabolic and Pro-Inflammatory Markers of Osteoarthritis and Favor Collagen Production and Metabolic Activity in Equine Articular Chondrocyte Organoids.

Articular cartilage experiences mechanical constraints leading to chondral defects that inevitably evolve into osteoarthritis (OA), because cartilage has poor intrinsic repair capacity. Although OA is an incurable degenerative disease, several dietary supplements may help improve OA outcomes. In this study, we investigated the effects of Dielen® hydrolyzed fish collagens from skin (Promerim®30 and Promerim®60) and cartilage (Promerim®40) to analyze the phenotype and metabolism of equine articular chondrocytes (eACs) cultured as organoids. Here, our findings demonstrated the absence of cytotoxicity and the beneficial effect of Promerim® hydrolysates on eAC metabolic activity under physioxia; further, Promerim®30 also delayed eAC senescence. To assess the effect of Promerim® in a cartilage-like tissue, eACs were cultured as organoids under hypoxia with or without BMP-2 and/or IL-1ß. In some instances, alone or in the presence of IL-1ß, Promerim®30 and Promerim®40 increased protein synthesis of collagen types I and II, while decreasing transcript levels of proteases involved in OA pathogenesis, namely Htra1, and the metalloproteinases Mmp1-3, Adamts5, and Cox2. Both Promerim® hydrolysates also decreased Htra1 protein amounts, particularly in inflammatory conditions. The effect of Promerim® was enhanced under inflammatory conditions, possibly due to a decrease in the synthesis of inflammation-associated molecules. Finally, Promerim® favored in vitro repair in a scratch wound assay through an increase in cell proliferation or migration. Altogether, these data show that Promerim®30 and 40 hold promise as dietary supplements to relieve OA symptoms in patients and to delay OA progression.

Marine Collagen Hydrolysates Promote Collagen Synthesis, Viability and Proliferation While Downregulating the Synthesis of Pro-Catabolic Markers in Human Articular Chondrocytes.

Cartilage is a non-innervated and non-vascularized tissue. It is composed of one main cell type, the chondrocyte, which governs homeostasis within the cartilage tissue, but has low metabolic activity. Articular cartilage undergoes substantial stresses that lead to chondral defects, and inevitably osteoarthritis (OA) due to the low intrinsic repair capacity of cartilage. OA remains an incurable degenerative disease. In this context, several dietary supplements have shown promising results, notably in the relief of OA symptoms. In this study, we investigated the effects of collagen hydrolysates derived from fish skin (Promerim®30 and Promerim®60) and fish cartilage (Promerim®40) on the phenotype and metabolism of human articular chondrocytes (HACs). First, we demonstrated the safety of Promerim® hydrolysates on HACs cultured in monolayers. Then we showed that, Promerim® hydrolysates can increase the HAC viability and proliferation, while decreasing HAC SA-ß-galactosidase activity. To evaluate the effect of Promerim® on a more relevant model of culture, HAC were cultured as organoids in the presence of Promerim® hydrolysates with or without IL-1ß to mimic an OA environment. In such conditions, Promerim® hydrolysates led to a decrease in the transcript levels of some proteases that play a major role in the development of OA, such as Htra1 and metalloproteinase-1. Promerim® hydrolysates downregulated HtrA1 protein expression. In contrast, the treatment of cartilage organoids with Promerim® hydrolysates increased the neosynthesis of type I collagen (Promerim®30, 40 and 60) and type II collagen isoforms (Promerim®30 and 40), the latter being the major characteristic component of the cartilage extracellular matrix. Altogether, our results demonstrate that the use of Promerim® hydrolysates hold promise as complementary dietary supplements in combination with the current classical treatments or as a preventive therapy to delay the occurrence of OA in humans.

Evaluation of Allogeneic Bone-Marrow-Derived and Umbilical Cord Blood-Derived Mesenchymal Stem Cells to Prevent the Development of Osteoarthritis in An Equine Model.

Osteoarthritis (OA) is a significant cause of pain in both humans and horses with a high socio-economic impact. The horse is recognized as a pertinent model for human OA. In both species, regenerative therapy with allogeneic mesenchymal stem cells (MSCs) appears to be a promising treatment but, to date, no in vivo studies have attempted to compare the effects of different cell sources on the same individuals. The objective of this study is to evaluate the ability of a single blinded intra-articular injection of allogeneic bone-marrow (BM) derived MSCs and umbilical cord blood (UCB) derived MSC to limit the development of OA-associated pathological changes compared to placebo in a post-traumatic OA model applied to all four fetlock joints of eight horses. The effect of the tissue source (BM vs. UCB) is also assessed on the same individuals. Observations were carried out using clinical, radiographic, ultrasonographic, and magnetic resonance imaging methods as well as biochemical analysis of synovial fluid and postmortem microscopic and macroscopic evaluations of the joints until Week 12. A significant reduction in the progression of OA-associated changes measured with imaging techniques, especially radiography, was observed after injection of bone-marrow derived mesenchymal stem cells (BM-MSCs) compared to contralateral placebo injections. These results indicate that allogeneic BM-MSCs are a promising treatment for OA in horses and reinforce the importance of continuing research to validate these results and find innovative strategies that will optimize the therapeutic potential of these cells. However, they should be considered with caution given the low number of units per group.

Tumor Suppressive Role of miR-342-5p in Human Chondrosarcoma Cells and 3D Organoids.

Chondrosarcomas are malignant bone tumors. Their abundant cartilage-like extracellular matrix and their hypoxic microenvironment contribute to their resistance to chemotherapy and radiotherapy, and no effective therapy is currently available. MicroRNAs (miRNAs) may be an interesting alternative in the development of therapeutic options. Here, for the first time in chondrosarcoma cells, we carried out high-throughput functional screening using impedancemetry, and identified five miRNAs with potential antiproliferative or chemosensitive effects on SW1353 chondrosarcoma cells. The cytotoxic effects of miR-342-5p and miR-491-5p were confirmed on three chondrosarcoma cell lines, using functional validation under normoxia and hypoxia. Both miRNAs induced apoptosis and miR-342-5p also induced autophagy. Western blots and luciferase reporter assays identified for the first time Bcl-2 as a direct target of miR-342-5p, and also Bcl-xL as a direct target of both miR-342-5p and miR-491-5p in chondrosarcoma cells. MiR-491-5p also inhibited EGFR expression. Finally, only miR-342-5p induced cell death on a relevant 3D chondrosarcoma organoid model under hypoxia that mimics the in vivo microenvironment. Altogether, our results revealed the tumor suppressive activity of miR-342-5p, and to a lesser extent of miR-491-5p, on chondrosarcoma lines. Through this study, we also confirmed the potential of Bcl-2 family members as therapeutic targets in chondrosarcomas.

Chondrogenic Differentiation of Defined Equine Mesenchymal Stem Cells Derived from Umbilical Cord Blood for Use in Cartilage Repair Therapy.

Cartilage engineering is a new strategy for the treatment of cartilage damage due to osteoarthritis or trauma in humans. Racehorses are exposed to the same type of cartilage damage and the anatomical, cellular, and biochemical properties of their cartilage are comparable to those of human cartilage, making the horse an excellent model for the development of cartilage engineering. Human mesenchymal stem cells (MSCs) differentiated into chondrocytes with chondrogenic factors in a biomaterial appears to be a promising therapeutic approach for direct implantation and cartilage repair. Here, we characterized equine umbilical cord blood-derived MSCs (eUCB-MSCs) and evaluated their potential for chondrocyte differentiation for use in cartilage repair therapy. Our results show that isolated eUCB-MSCs had high proliferative capacity and differentiated easily into osteoblasts and chondrocytes, but not into adipocytes. A three-dimensional (3D) culture approach with the chondrogenic factors BMP-2 and TGF-ß1 potentiated chondrogenic differentiation with a significant increase in cartilage-specific markers at the mRNA level (Col2a1, Acan, Snorc) and the protein level (type II and IIB collagen) without an increase in hypertrophic chondrocyte markers (Col10a1 and Mmp13) in normoxia and in hypoxia. However, these chondrogenic factors caused an increase in type I collagen, which can be reduced using small interfering RNA targeting Col1a2. This study provides robust data on MSCs characterization and demonstrates that eUCB-MSCs have a great potential for cartilage tissue engineering.

Improvement of the Chondrocyte-Specific Phenotype upon Equine Bone Marrow Mesenchymal Stem Cell Differentiation: Influence of Culture Time, Transforming Growth Factors and Type I Collagen siRNAs on the Differentiation Index.

Articular cartilage is a tissue characterized by its poor intrinsic capacity for self-repair. This tissue is frequently altered upon trauma or in osteoarthritis (OA), a degenerative disease that is currently incurable. Similar musculoskeletal disorders also affect horses and OA incurs considerable economic loss for the equine sector. In the view to develop new therapies for humans and horses, significant progress in tissue engineering has led to the emergence of new generations of cartilage therapy. Matrix-associated autologous chondrocyte implantation is an advanced 3D cell-based therapy that holds promise for cartilage repair. This study aims to improve the autologous chondrocyte implantation technique by using equine mesenchymal stem cells (MSCs) from bone marrow differentiated into chondrocytes that can be implanted in the chondral lesion. The optimized protocol relies on culture under hypoxia within type I/III collagen sponges. Here, we explored three parameters that influence MSC differentiation: culture times, growth factors and RNA interference strategies. Our results suggest first that an increase in culture time from 14 to 28 or 42 days lead to a sharp increase in the expression of chondrocyte markers, notably type II collagen (especially the IIB isoform), along with a concomitant decrease in HtrA1 expression. Nevertheless, the expression of type I collagen also increased with longer culture times. Second, regarding the growth factor cocktail, TGF-ß3 alone showed promising result but the previously tested association of BMP-2 and TGF-ß1 better limits the expression of type I collagen. Third, RNA interference targeting Col1a2 as well as Col1a1 mRNA led to a more significant knockdown, compared with a conventional strategy targeting Col1a1 alone. This chondrogenic differentiation strategy showed a strong increase in the Col2a1:Col1a1 mRNA ratio in the chondrocytes derived from equine bone marrow MSCs, this ratio being considered as an index of the functionality of cartilage. These data provide evidence of a more stable chondrocyte phenotype when combining Col1a1 and Col1a2 siRNAs associated to a longer culture time in the presence of BMP-2 and TGF-ß1, opening new opportunities for preclinical trials in the horse. In addition, because the horse is an excellent model for human articular cartilage disorders, the equine therapeutic approach developed here can also serve as a preclinical step for human medicine.

RNA Interference and BMP-2 Stimulation Allows Equine Chondrocytes Redifferentiation in 3D-Hypoxia Cell Culture Model: Application for Matrix-Induced Autologous Chondrocyte Implantation.

As in humans, osteoarthritis (OA) causes considerable economic loss to the equine industry. New hopes for cartilage repair have emerged with the matrix-associated autologous chondrocyte implantation (MACI). Nevertheless, its limitation is due to the dedifferentiation occurring during the chondrocyte amplification phase, leading to the loss of its capacity to produce a hyaline extracellular matrix (ECM). To enhance the MACI therapy efficiency, we have developed a strategy for chondrocyte redifferentiation, and demonstrated its feasibility in the equine model. Thus, to mimic the cartilage microenvironment, the equine dedifferentiated chondrocytes were cultured in type I/III collagen sponges for 7 days under hypoxia in the presence of BMP-2. In addition, chondrocytes were transfected by siRNA targeting Col1a1 and Htra1 mRNAs, which are overexpressed during dedifferentiation and OA. To investigate the quality of the neo-synthesized ECM, specific and atypical cartilage markers were evaluated by RT-qPCR and Western blot. Our results show that the combination of 3D hypoxia cell culture, BMP-2 (Bone morphogenetic protein-2), and RNA interference, increases the chondrocytes functional indexes (Col2a1/Col1a1, Acan/Col1a1), leading to an effective chondrocyte redifferentiation. These data represent a proof of concept for this process of application, in vitro, in the equine model, and will lead to the improvement of the MACI efficiency for cartilage tissue engineering therapy in preclinical/clinical trials, both in equine and human medicine.

Hypoxia Is a Critical Parameter for Chondrogenic Differentiation of Human Umbilical Cord Blood Mesenchymal Stem Cells in Type I/III Collagen Sponges.

Umbilical cord blood (UCB) is an attractive alternative to bone marrow for isolation of mesenchymal stem cells (MSCs) to treat articular cartilage defects. Here, we set out to determine the growth factors (bone morphogenetic protein 2 (BMP-2) and transforming growth factor-ß (TGF-ß1)) and oxygen tension effects during chondrogenesis of human UCB-MSCs for cartilage engineering. Chondrogenic differentiation was induced using 3D cultures in type I/III collagen sponges with chondrogenic factors in normoxia (21% O2) or hypoxia (<5% O2) for 7, 14 and 21 days. Our results show that UCB-MSCs can be committed to chondrogenesis in the presence of BMP-2+TGF-ß1. Normoxia induced the highest levels of chondrocyte-specific markers. However, hypoxia exerted more benefit by decreasing collagen X and matrix metalloproteinase-13 (MMP13) expression, two chondrocyte hypertrophy markers. However, a better chondrogenesis was obtained by switching oxygen conditions, with seven days in normoxia followed by 14 days in hypoxia, since these conditions avoid hypertrophy of hUCB-MSC-derived chondrocytes while maintaining the expression of chondrocyte-specific markers observed in normoxia. Our study demonstrates that oxygen tension is a key factor for chondrogenesis and suggests that UBC-MSCs 3D-culture should begin in normoxia to obtain a more efficient chondrocyte differentiation before placing them in hypoxia for chondrocyte phenotype stabilization. UCB-MSCs are therefore a reliable source for cartilage engineering.

Cartilage tissue engineering: molecular control of chondrocyte differentiation for proper cartilage matrix reconstruction.

BACKGROUND: Articular cartilage defects are a veritable therapeutic problem because therapeutic options are very scarce. Due to the poor self-regeneration capacity of cartilage, minor cartilage defects often lead to osteoarthritis. Several surgical strategies have been developed to repair damaged cartilage. Autologous chondrocyte implantation (ACI) gives encouraging results, but this cell-based therapy involves a step of chondrocyte expansion in a monolayer, which results in the loss in the differentiated phenotype. Thus, despite improvement in the quality of life for patients, reconstructed cartilage is in fact fibrocartilage. Successful ACI, according to the particular physiology of chondrocytes in vitro, requires active and phenotypically stabilized chondrocytes. SCOPE OF REVIEW: This review describes the unique physiology of cartilage, with the factors involved in its formation, stabilization and degradation. Then, we focus on some of the most recent advances in cell therapy and tissue engineering that open up interesting perspectives for maintaining or obtaining the chondrogenic character of cells in order to treat cartilage lesions. MAJOR CONCLUSIONS: Current research involves the use of chondrocytes or progenitor stem cells, associated with "smart" biomaterials and growth factors. Other influential factors, such as cell sources, oxygen pressure and mechanical strain are considered, as are recent developments in gene therapy to control the chondrocyte differentiation/dedifferentiation process. GENERAL SIGNIFICANCE: This review provides new information on the mechanisms regulating the state of differentiation of chondrocytes and the chondrogenesis of mesenchymal stem cells that will lead to the development of new restorative cell therapy approaches in humans. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.

In vitro engineering of human 3D chondrosarcoma: a preclinical model relevant for investigations of radiation quality impact.

BACKGROUND: The benefit of better ballistic and higher efficiency of carbon ions for cancer treatment (hadron-therapy) is asserted since decades, especially for unresectable or resistant tumors like sarcomas. However, hadron-therapy with carbon ions stays underused and raises some concerns about potential side effects for patients. Chondrosarcoma is a cartilaginous tumor, chemo- and radiation-resistant, that lacks reference models for basic and pre-clinical studies in radiation-biology. Most studies about cellular effects of ionizing radiation, including hadrons, were performed under growth conditions dramatically different from human homeostasis. Tridimensional in vitro models are a fair alternative to animal models to approach tissue and tumors microenvironment. METHODS: By using a collagen matrix, standardized culture conditions, physiological oxygen tension and a well defined chondrosarcoma cell line, we developed a pertinent in vitro 3D model for hadron-biology studies. Low- and high-Linear Energy Transfer (LET) ionizing radiations from GANIL facilities of ~1 keV/µm and 103 ± 4 keV/µm were used respectively, at 2 Gy single dose. The impact of radiation quality on chondrosarcoma cells cultivated in 3D was analyzed on cell death, cell proliferation and DNA repair. RESULTS: A fair distribution of chondrosarcoma cells was observed in the whole 3D scaffold. Moreover, LET distribution in depth, for ions, was calculated and found acceptable for radiation-biology studies using this kind of scaffold. No difference in cell toxicity was observed between low- and high-LET radiations but a higher rate of proliferation was displayed following high-LET irradiation. Furthermore, 3D models presented a higher and longer induction of H2AX phosphorylation after 2 Gy of high-LET compared to low-LET radiations. CONCLUSIONS: The presented results show the feasibility and usefulness of our 3D chondrosarcoma model in the study of the impact of radiation quality on cell fate. The observed changes in our tissue-like model after ionizing radiation exposure may explain some discrepancies between radiation-biology studies and clinical data.

The p65 subunit of NF-κB inhibits COL1A1 gene transcription in human dermal and scleroderma fibroblasts through its recruitment on promoter by protein interaction with transcriptional activators (c-Krox, Sp1, and Sp3).

Transcriptional mechanisms regulating type I collagen genes expression in physiopathological situations are not completely known. In this study, we have investigated the role of nuclear factor-κB (NF-κB) transcription factor on type I collagen expression in adult normal human (ANF) and scleroderma (SF) fibroblasts. We demonstrated that NF-κB, a master transcription factor playing a major role in immune response/apoptosis, down-regulates COL1A1 expression by a transcriptional control involving the -112/-61 bp sequence. This 51-bp region mediates the action of two zinc fingers, Sp1 (specific protein-1) and Sp3, acting as trans-activators of type I collagen expression in ANF and SF. Knockdown of each one of these trans factors by siRNA confirmed the trans-activating effect of Sp1/Sp3 and the p65 subunit of NF-κB trans-inhibiting effect on COL1A1 expression. Despite no existing κB consensus sequence in the COL1A1 promoter, we found that Sp1/Sp3/c-Krox and NF-κB bind and/or are recruited on the proximal promoter in chromatin immunoprecipitation (ChIP) assays. Attempts to elucidate whether interactions between Sp1/Sp3/c-Krox and p65 are necessary to mediate the NF-κB inhibitory effect on COL1A1 in ANF and SF were carried out; in this regard, immunoprecipitation assays revealed that they interact, and this was validated by re-ChIP. Finally, the knockdown of Sp1/Sp3/c-Krox prevents the p65 inhibitory effect on COL1A1 transcription in ANF, whereas only the siRNAs targeting Sp3 and c-Krox provoked the same effect in SF, suggesting that particular interactions are characteristic of the scleroderma phenotype. In conclusion, our findings highlight a new mechanism for COL1A1 transcriptional regulation by NF-κB, and these data could allow the development of new antifibrotic strategies.

Regulatory mechanism of transforming growth factor beta receptor type II degradation by interleukin-1 in primary chondrocytes.

Interleukin-1ß (IL-1ß), a key-cytokine in osteoarthritis, impairs TGFß signaling through TßRII down-regulation by increasing its degradation. Here, we investigated the molecular mechanism that controls TßRII fate in IL-1ß treated cells. Chondrocytes were treated with IL-1ß in the presence of different inhibitors. TßRII and Cav-1 expression were assayed by Western blot and RT-PCR. We showed that IL-1ß-induced degradation of TßRII is dependent on proteasome and on its internalization in caveolae. In addition, IL-1ß enhances Cav-1 expression, a major constituent of lipid raft. In conclusion, we enlighten a new mechanism by which IL-1ß antagonizes TGFß pathway and propose a model of TßRII turnover regulation upon IL-1ß treatment.

Equine osteoarthritis: Strategies to enhance mesenchymal stromal cell-based acellular therapies.

Osteoarthritis (OA) is a degenerative disease that eventually leads to the complete degradation of articular cartilage. Articular cartilage has limited intrinsic capacity for self-repair and, to date, there is no curative treatment for OA. Humans and horses have a similar articular cartilage and OA etiology. Thus, in the context of a One Health approach, progress in the treatment of equine OA can help improve horse health and can also constitute preclinical studies for human medicine. Furthermore, equine OA affects horse welfare and leads to significant financial losses in the equine industry. In the last few years, the immunomodulatory and cartilage regenerative potentials of mesenchymal stromal cells (MSCs) have been demonstrated, but have also raised several concerns. However, most of MSC therapeutic properties are contained in their secretome, particularly in their extracellular vesicles (EVs), a promising avenue for acellular therapy. From tissue origin to in vitro culture methods, various aspects must be taken into consideration to optimize MSC secretome potential for OA treatment. Immunomodulatory and regenerative properties of MSCs can also be enhanced by recreating a pro-inflammatory environment to mimic an in vivo pathological setting, but more unusual methods also deserve to be investigated. Altogether, these strategies hold substantial potential for the development of MSC secretome-based therapies suitable for OA management. The aim of this mini review is to survey the most recent advances on MSC secretome research with regard to equine OA.

Effect of pro-inflammatory cytokine priming and storage temperature of the mesenchymal stromal cell (MSC) secretome on equine articular chondrocytes.

Context: Osteoarthritis (OA) is an invalidating articular disease characterized by cartilage degradation and inflammatory events. In horses, OA is associated with up to 60% of lameness and leads to reduced animal welfare along with extensive economic losses; currently, there are no curative therapies to treat OA. The mesenchymal stromal cell (MSC) secretome exhibits anti-inflammatory properties, making it an attractive candidate for improving the management of OA. In this study, we determined the best storage conditions for conditioned media (CMs) and tested whether priming MSCs with cytokines can enhance the properties of the MSC secretome. Methods: First, properties of CMs collected from bone-marrow MSC cultures and stored at -80°C, -20°C, 4°C, 20°C or 37°C were assessed on 3D cultures of equine articular chondrocytes (eACs). Second, we primed MSCs with IL-1ß, TNF-α or IFN-γ, and evaluated the MSC transcript levels of immunomodulatory effectors and growth factors. The primed CMs were also harvested for subsequent treatment of eACs, either cultured in monolayers or as 3D cell cultures. Finally, we evaluated the effect of CMs on the proliferation and the phenotype of eACs and the quality of the extracellular matrix of the neosynthesized cartilage. Results: CM storage at -80°C, -20°C, and 4°C improved collagen protein accumulation, cell proliferation and the downregulation of inflammation. The three cytokines chosen for the MSC priming influenced MSC immunomodulator gene expression, although each cytokine led to a different pattern of MSC immunomodulation. The cytokine-primed CM had no major effect on eAC proliferation, with IL-1ß and TNF-α slightly increasing collagen (types IIB and I) accumulation in eAC 3D cultures (particularly with the CM derived from MSCs primed with IL-1ß), and IFN-γ leading to a marked decrease. IL-1ß-primed CMs resulted in increased eAC transcript levels of MMP1, MMP13 and HTRA1, whereas IFNγ-primed CMs decreased the levels of HTRA1 and MMP13. Conclusion: Although the three cytokines differentially affected the expression of immunomodulatory molecules, primed CMs induced a distinct effect on eACs according to the cytokine used for MSC priming. Different mechanisms seemed to be triggered by each priming cytokine, highlighting the need for further investigation. Nevertheless, this study demonstrates the potential of MSC-CMs for improving equine OA management.

MiR-4270 acts as a tumor suppressor by directly targeting Bcl-xL in human osteosarcoma cells.

Chondrosarcomas and osteosarcomas are malignant bone tumors with a poor prognosis when unresectable or metastasized. Moreover, radiotherapy and chemotherapy could be ineffective. MiRNAs represent an alternative therapeutic approach. Based on high-throughput functional screening, we identified four miRNAs with a potential antiproliferative effect on SW1353 chondrosarcoma cells. Individual functional validations were then performed in SW1353 cells, as well as in three osteosarcoma cell lines. The antiproliferative and cytotoxic effects of miRNAs were evaluated in comparison with a positive control, miR-342-5p. The cytotoxic effect of four selected miRNAs was not confirmed on SW1353 cells, but we unambiguously revealed that miR-4270 had a potent cytotoxic effect on HOS and MG-63 osteosarcoma cell lines, but not on SaOS-2 cell line. Furthermore, like miR-342-5p, miR-4270 induced apoptosis in these two cell lines. In addition, we provided the first report of Bcl-xL as a direct target of miR-4270. MiR-4270 also decreased the expression of the anti-apoptotic protein Mcl-1, and increased the expression of the pro-apoptotic protein Bak. Our findings demonstrated that miR-4270 has tumor suppressive activity in osteosarcoma cells, particularly through Bcl-xL downregulation.

Mechanisms involved in cardioprotective effects of pravastatin administered during reoxygenation in human myocardium in vitro.

BACKGROUND: The authors investigated the effect of pravastatin during reoxygenation after myocardial hypoxia and examined the involvement of nitric oxide synthase, mitochondrial permeability transition pore, and expression of markers of apoptosis in human myocardium in vitro. METHODS: Human atrial trabeculae were exposed to hypoxia for 30 min and reoxygenation for 60 min (control group; n = 10). Pravastatin (5, 10, 50, 75 µM; n = 6 in each group) was administered throughout the reoxygenation. In separate groups (n = 6 in each group), pravastatin 50 µM was administered in the presence of 200 µM L-NG-nitroarginine methyl ester, a nitric oxide synthase inhibitor, and 50 µM atractyloside, the mitochondrial permeability transition pore opener. The primary endpoint was the developed force of contraction at the end of reoxygenation, expressed as a percentage of baseline (mean ± SD). Protein expression of BAD, phospho-BAD, caspase 3, Pim-1 kinase, and Bcl-2 were measured using Western immunoblotting. RESULTS: The administration of 10 (77 ± 5% of baseline), 50 (86 ± 6%), and 75 µM (88 ± 13%) pravastatin improved the force of contraction at the end of reoxygenation, compared with that of the control group (49 ± 11%; P < 0.001). These beneficial effects were prevented by L-NG-nitroarginine methyl ester and atractyloside. Compared with control group, the administration of 5 µM pravastatin did not modify the force of contraction. Pravastatin increased the phosphorylation of BAD, activated the expression of Pim-1 kinase and Bcl-2, and maintained the caspase 3 concentration relative to that of the respective untreated controls. CONCLUSIONS: Pravastatin, administered at reoxygenation, protected the human myocardium by preventing the mitochondrial permeability transition pore opening, phosphorylating BAD, activating nitric oxide synthase, Pim-1 kinase, and Bcl-2, and preserving the myocardium against the caspase 3 activation.