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Supplemental material, sj-docx-1-pih-10.1177_09544119221109019 for A preliminary study exploring the mechanical properties of normal and Mgp-deficient mouse femurs during early growth by Cédric Laurent, Alexandre Marano, Adrien Baldit, Maude Ferrari, Jean-Christophe Perrin, Olivier Perroud, Arnaud Bianchi and Hervé Kempf in Proceedings of the Institution of Mechanical Engineers, Part H: Journal of Engineering in Medicine

“Extracellular vesicles” (EVs) is a term gathering biological particles released from cells that act as messengers for cell-to-cell communication. Like cells, EVs have a membrane with a lipid bilayer, but unlike these latter, they have no nucleus and consequently cannot replicate. Several EV subtypes (e.g., exosomes, microvesicles) are described in the literature. However, the remaining lack of consensus on their specific markers prevents sometimes the full knowledge of their biogenesis pathway, causing the authors to focus on their biological effects and not their origins. EV signals depend on their cargo, which can be naturally sourced or altered (e.g., cell engineering). The ability for regeneration of adult articular cartilage is limited because this avascular tissue is partly made of chondrocytes with a poor proliferation rate and migration capacity. Mesenchymal stem cells (MSCs) had been extensively used in numerous in vitro and preclinical animal models for cartilage regeneration, and it has been demonstrated that their therapeutic effects are due to paracrine mechanisms involving EVs. Hence, using MSC-derived EVs as cell-free therapy tools has become a new therapeutic approach to improve regenerative medicine. EV-based therapy seems to show similar cartilage regenerative potential compared with stem cell transplantation without the associated hindrances (e.g., chromosomal aberrations, immunogenicity). The aim of this short review is to take stock of occurring EV-based treatments for cartilage regeneration according to their healing effects. The article focuses on cartilage regeneration through various sources used to isolate EVs (mature or stem cells among others) and beneficial effects depending on cargos produced from natural or tuned EVs.

To promote the nutritional and pharmacological values of four sea cucumber species (Holothuria poli, H. tubulosa, H. arguinensis, and H. sanctori), harvested from the Algerian coast, we aimed to study their proximate composition, fatty acid profile and angiotensin-converting enzyme (ACE) inhibitory activity. Their phospholipids were also used to elaborate nanoliposomes and to encapsulate peptides obtained from the same source. After the physico-chemical characterization of nanoliposomes and peptides, in vitro analyses were realized. The four holothurian species showed a high amount of protein (49.26–69.34%), and an impressive lipid profile of 27 fatty acids, mainly composed of polar fatty acids (91.16–93.85%), with a high polyunsaturated fatty acids (PUFA) content (50.90–71.80%), particularly eicosapentaenoic acid (EPA) (5.07–8.76%) and docosahexaenoic acid (DHA) (4.86–7.25%). A high phospholipids amount was also found (55.20–69.85%), mainly composed of phosphatidylcholine (PC) (51.48–58.56%). Their peptide fractions exhibited a high ACE inhibitory activity (IC50 0.30 to 0.51 mg/mL). The results also showed that the nanoliposomes do not induce cytotoxicity and cell death in human MSCs and no perturbation of proliferation for all the times and the tested concentrations, as well as the combined nanoliposomes and hydrolysates (HTS) at a concentration of 0.1 mg/mL. All four sea cucumbers show potential as a new source for omega-3, omega-6, and bioactive peptides.

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International audience; During skeletal mineralization, the sodium-phosphate co-transporter PiT1Slc20a1 is assumed to meet the phosphate requirements of bone-forming cells, although evidence is missing. Here, we used a conditional gene deletion approach to determine the role of PiT1 in growth plate chondrocytes. We show that PiT1 ablation shortly after birth generates a rapid and massive cell death in the center of the growth plate, together with an uncompensated ER stress, characterized by morphological changes and increased Chop, Atf4 and Bip expression. PiT1 expression in chondrocytes was not found at the cell membrane but co-localized with the ER marker ERp46, and was up-regulated by the unfolded protein response cascade. In addition, we identified the protein disulfide isomerase (Pdi) ER chaperone as a PiT1 binding partner and showed that PiT1 ablation impaired Pdi reductase activity. The ER stress induced by PiT1 deficiency in chondrocytes was associated with intracellular retention of aggrecan and Vegf-A, which was rescued by over-expressing a phosphate transport-deficient mutant of PiT1. Our data thus reveal a novel, Pi-transport independent function of PiT1, as a critical modulator of ER homeostasis and chondrocyte survival during endochondral ossification. This article is protected by copyright. All rights reserved.

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Microsomal prostaglandin E synthase (mPGES)-1 is a newly identified inducible enzyme of the arachidonic acid cascade with a key function in prostaglandin (PG)E2 synthesis. We investigated the kinetics of inducible cyclo-oxygenase (COX)-2 and mPGES-1 expression with respect to the production of 6-keto-PGF1alpha and PGE2 in rat chondrocytes stimulated with 10 ng/ml IL-1beta, and compared their modulation by peroxisome-proliferator-activated receptor (PPAR)gamma agonists. Real-time PCR analysis showed that IL-1beta induced COX-2 expression maximally (37-fold) at 12 hours and mPGES-1 expression maximally (68-fold) at 24 hours. Levels of 6-keto-PGF1alpha and PGE2 peaked 24 hours after stimulation with IL-1beta; the induction of PGE2 was greater (11-fold versus 70-fold, respectively). The cyclopentenone 15-deoxy-Delta12,14prostaglandin J2 (15d-PGJ2) decreased prostaglandin synthesis in a dose-dependent manner (0.1 to 10 microM), with more potency on PGE2 level than on 6-keto-PGF1alpha level (-90% versus -66% at 10 microM). A high dose of 15d-PGJ2 partly decreased COX-2 expression but decreased mPGES-1 expression almost completely at both the mRNA and protein levels. Rosiglitazone was poorly effective on these parameters even at 10 microM. Inhibitory effects of 10 microM 15d-PGJ2 were neither reduced by PPARgamma blockade with GW-9662 nor enhanced by PPARgamma overexpression, supporting a PPARgamma-independent mechanism. EMSA and TransAM analyses demonstrated that mutated IkappaBalpha almost completely suppressed the stimulating effect of IL-1beta on mPGES-1 expression and PGE2 production, whereas 15d-PGJ2 inhibited NF-kappaB transactivation. These data demonstrate the following in IL-1-stimulated rat chondrocytes: first, mPGES-1 is rate limiting for PGE2 synthesis; second, activation of the prostaglandin cascade requires NF-kappaB activation; third, 15d-PGJ2 strongly inhibits the synthesis of prostaglandins, in contrast with rosiglitazone; fourth, inhibition by 15d-PGJ2 occurs independently of PPARgamma through inhibition of the NF-kappaB pathway; fifth, mPGES-1 is the main target of 15d-PGJ2.

Background and Purpose. We investigated the potency of Peroxisome Proliferators-Activated Receptors (PPARs) α, β/δ, and γ agonists to modulate Transforming Growth Factor-β1 (TGF-β1-) induced collagen production or changes in Tissue Inhibitor of Matrix Metalloproteinase- (TIMP-) 1/Matrix Metalloproteinase (MMP) balance in rat chondrocytes embedded in alginate beads. Experimental Approach. Collagen production was evaluated by quantitative Sirius red staining, while TIMP-1 protein levels and global MMP (-1, -2, -3, -7, and -9) or specific MMP-13 activities were measured by ELISA and fluorigenic assays in culture media, respectively. Levels of mRNA for type II collagen, TIMP-1, and MMP-3 & 13 were quantified by real-time PCR. Key Results. TGF-β1 increased collagen deposition and type II collagen mRNA levels, while inducing TIMP-1 mRNA and protein expression. In contrast, it decreased global MMP or specific MMP-13 activities, while decreasing MMP-3 or MMP-13 mRNA levels. PPAR agonists reduced most of the effects of TGF-β1 on changes in collagen metabolism and TIMP-1/MMP balance in rat in a PPAR-dependent manner, excepted for Wy14643 on MMP activities. Conclusions and Implications. PPAR agonists reduce TGF-β1-modulated ECM turnover and inhibit chondrocyte activities crucial for collagen biosynthesis, and display a different inhibitory profile depending on selectivity for PPAR isotypes.

International audience; Peroxisome proliferators activated receptors (PPAR) are ligand-inducible nuclear transacting factors comprising three subtypes, PPARα, PPARβ/δ and PPARγ, which play a key role in lipids and glucose homeostasis. All PPAR subtypes have been identified in joint or inflammatory cells and their activation resulted in a transcriptional repression of pro-inflammatory cytokines (IL-1, TNFα), early inflammatory genes (NOS2, COX-2, mPGES-1) or matrix metalloproteases (MMP-1, MMP-13), at least for the γ subtype. PPAR full agonists were also shown to stimulate IL-1 receptor antagonist (IL-1Ra) production by cytokine-stimulated articular cells in a subtype-dependent manner. These anti-inflammatory and anti-catabolic properties were confirmed in animal models of joint diseases where PPAR agonists reduced synovial inflammation while preventing cartilage destruction or inflammatory bone loss, although many effects required much higher doses than needed to restore insulin sensitivity or to lower circulating lipid levels. However, these promising effects of PPAR full agonists were hampered by their ability to reduce the growth factor-dependent synthesis of extracellular matrix components or to induce chondrocyte apoptosis, by the possible contribution of immunosuppressive properties to their anti-arthritic effects, by the increased adipocyte differentiation secondary to prolonged stimulation of PPARγ, and by a variable contribution of PPAR subtypes depending on the system. Clinical data are scarce in rheumatoid arthritis (RA) patients whereas thousands of patients worldwilde, treated with PPAR agonists for type 2 diabetes or dyslipidemia, are paradoxically prone to suffer from osteoarthritis (OA). Whereas high dosage of full agonists may expose RA patients to cardiovascular adverse effects, the proof of concept that PPAR agonists have therapeutical relevance to OA may benefit from an epidemiological follow-up of joint lesions in diabetic or hyperlipidemic patients treated for long periods of time with glitazones or fibrates. Additionally, cellular and animal studies are required to assess whether partial agonists of PPAR (SPPARMs) may preserve therapeutical properties with potentially less safety concern.; Les récepteurs activés par les proliférateurs de peroxysomes (PPAR) sont des facteurs de transcription inductibles par des ligands dont il existe trois sous-types, PPARα, PPARβ/δ et PPARγ jouant un rôle primordial dans le métabolisme des lipides et l'homéostasie du glucose. Ces trois sous-types sont également exprimés dans les cellules résidentes de l'articulation et celles de l'infiltrat inflammatoire, et leur activation réprime l'expression de cytokines pro-inflammatoires (IL-1, TNFα), de gènes précoces de l'inflammation (iNOS, COX-2, mPGES-1) ou de métalloprotéases matricielles (MMP-1, MMP-13), tout du moins pour le sous-type γ. Les agonistes de PPAR sont également capables de stimuler la production de l'antagoniste du récepteur à l'IL-1 (IL-1Ra) par les cellules articulaires soumises à un stimulus cytokinique. Ces potentialités anti-inflammatoires et anti-cataboliques ont été confirmées dans des modèles animaux d'arthropathie, dans lesquels les agonistes de PPAR diminuent l'inflammation synoviale et préviennent la destruction du cartilage ou la perte osseuse inflammatoire, même si les posologies requises sont très supérieures à celles permettant de normaliser les taux circulants de lipides ou de rétablir la sensibilité des tissus périphériques à l'insuline. Ces effets prometteurs des agonistes sont cependant pondérés par leur capacité à diminuer l'effet trophique des facteurs de croissance sur la biosynthèse des composants matriciels ou à provoquer une apoptose chondrocytaire, par leur effet immunosuppresseur responsable d'une surestimation des propriétés anti-inflammatoires dans les polyarthrites expérimentales, par le renforcement de la différenciation adipocytaire lors d'une stimulation prolongée de PPARγ, et par la contribution variable des sous-types selon le système expérimental. Les données cliniques restent anecdotiques dans la polyarthrite rhumatoïde alors que, paradoxalement, des milliers de patients qui sont traités quotidiennement par des agonistes de PPAR pour un diabète de type 2 ou une dyslipidémie, souffrent conjointement d'arthrose. Alors que les fortes posologies d'agonistes exposeraient les patients polyarthritiques à un risque important d'effets indésirables, notamment cardiovasculaires, la démonstration de l'intérêt des agonistes de PPAR dans l'arthrose pourrait être apportée par une étude épidémiologique de l'incidence ou de l'évolution de la maladie arthrosique chez les patients diabétiques et/ou dyslipidémiques traités par des glitazones ou des fibrates. Il sera également opportun d'étudier si des agonistes partiels de PPAR permettront de conserver certaines potentialités thérapeutiques tout en diminuant le risque d'effets indésirables.

International audience; Microsomal prostaglandin E synthase (mPGES)-1 is a newly identified inducible enzyme of the arachidonic acid cascade with a key function in prostaglandin (PG)E 2 synthesis. We investigated the kinetics of inducible cyclo-oxygenase (COX)-2 and mPGES-1 expression with respect to the production of 6-keto-PGF 1α and PGE 2 in rat chondrocytes stimulated with 10 ng/ml IL-1β, and compared their modulation by peroxisome-proliferator-activated receptor (PPAR)γ agonists. Real-time PCR analysis showed that IL-1β induced COX-2 expression maximally (37-fold) at 12 hours and mPGES-1 expression maximally (68-fold) at 24 hours. Levels of 6-keto-PGF 1α and PGE 2 peaked 24 hours after stimulation with IL-1β; the induction of PGE 2 was greater (11-fold versus 70-fold, respectively). The cyclopentenone 15-deoxy-∆ 12,14 prostaglandin J 2 (15d-PGJ 2) decreased prostaglandin synthesis in a dose-dependent manner (0.1 to 10 µM), with more potency on PGE 2 level than on 6-keto-PGF 1α level (-90% versus-66% at 10 µM). A high dose of 15d-PGJ 2 partly decreased COX-2 expression but decreased mPGES-1 expression almost completely at both the mRNA and protein levels. Rosiglitazone was poorly effective on these parameters even at 10 µM. Inhibitory effects of 10 µM 15d-PGJ 2 were neither reduced by PPARγ blockade with GW-9662 nor enhanced by PPARγ overexpression, supporting a PPARγ-independent mechanism. EMSA and TransAM ® analyses demonstrated that mutated IκBα almost completely suppressed the stimulating effect of IL-1β on mPGES-1 expression and PGE 2 production, whereas 15d-PGJ 2 inhibited NF-κB transactivation. These data demonstrate the following in IL-1-stimulated rat chondrocytes: first, mPGES-1 is rate limiting for PGE 2 synthesis; second, activation of the prostaglandin cascade requires NF-κB activation; third, 15d-PGJ 2 strongly inhibits the synthesis of prostaglandins, in contrast with rosiglitazone; fourth, inhibition by 15d-PGJ 2 occurs independently of PPARγ through inhibition of the NF-κB pathway; fifth, mPGES-1 is the main target of 15d-PGJ 2 .