- Participants will have access to novel information on cell-cell communication in cartilage lesions and during cartilage repair.
19.1.1 - The Role of Cartilage Oligomeric Matrix Protein (COMP) in Chondrocyte Behaviour and Osteoarthritis
Abstract
Introduction
The cartilage extracellular matrix (ECM) contains two suprastructures, a collagen meshwork and a proteoglycan gel, providing the tissue with both tensile strength and compressive resistance. These suprastructures are further stabilized by a large number of small proteoglycans and many non-collagenous glycoproteins, like e.g. several members of the thrombospondin family. The continuous degradation of the cartilage ECM is a hallmark of osteoarthritis (OA) and recent studies suggest that ECM proteins and proteolytic fragments released from degenerating cartilage may become biologically active factors and contribute to further joint destruction.Content
Thrombospondin-5, also referred to as cartilage oligomeric matrix protein (COMP), is ubiquitously expressed in healthy cartilage. In cartilage tissue, COMP interacts with collagens, proteoglycans and minor components (fibronectin, matrilins), most likely stabilizing the ECM. In addition to this structural function, COMP is involved in collagen secretion and acts as a catalyst for collagen fibril formation. Further, it has been shown that COMP can serve as a sensitive marker for the chondrocyte phenotype. In early OA, COMP is degraded but re-expressed at later stages of the disease. COMP is released from osteoarthritic cartilage and therefore widely used as diagnsotic OA biomarker. Indeed, serum levels correlate with both the number of affected joints and the severity of the disease. Using proteomic approaches and mass spectrometry, specific COMP-derived peptides were identified in OA cartilage but not in healthy tissue.However, the function of COMP in OA tissue as well as the contribution of COMP fragments to the progression of OA is still largely unknown. Therefore, we recombinantly expressed full-length COMP and fragments thereof in HEK293-EBNA cells and purified the proteins from supernatants via affinity chromatography. We treated human primary chondrocytes with these COMP constructs and analyzed the cellular response in detail. We investigated the chondrocyte proliferation, migration, adhesion, differentiation and ECM synthesis. In addition, the activation of specific intracellular signalling pathways was monitored using immunoblotting and the expression of inflammatory cytokines like interleukin-6 and TNF-α was investigated by PCR.
We could show that full-length COMP is able to attract chondrocytes and to mediate cell attachment but does not affect cell proliferation. COMP stimulates the expression of chondrocyte-specific matrix proteins like collagens II, IX and XII and contributes to the stabilization of the chondrocyte phenotype by increasing the ratio of collagen II to collagen I expression. Full-length COMP induces a time- and concentration-dependent phosporylation of the MAP kinases ERK1/2 while JNK and p38 kinase are not activated. COMP fragments seem to have only minor effects. Neither full length COMP nor its fragments increase the expression of inflammatory cytokines.
In summary, we could show that full-length COMP has beneficial effects on chondrocyte behavior and phenotype stabilization. Therefore, its re-expression in later OA stages might be interpreted as an attempt to counteract cartilage degeneration. COMP is degraded during OA progression but the degradation products seem to be rather inert. In summary, full-length COMP might be a promising candidate to be added in autologous chondrocyte implantation approaches or incorporated into scaffolds in cartilage tissue engineering.
19.1.2 - Mechanisms Underpinning Therapeutic Use of Mesenchymal Stromal Cells for Cartilage Protection in Osteoarthritis
Abstract
Introduction
Mesenchymal stromal cells (MSCs) are an emerging therapeutic tool for treatment of osteoarthritis (OA) with recent systematic reviews of early stage clinical trials for knee OA with data from over 500 patients indicating safety and some efficacy with modulation of pain1,2. Historically, the initial rationale for MSC use in osteoarthritis (OA) was based on their ability to undergo chondrogenic differentiation. However, we first demonstrated that MSCs do not engraft to the degenerating cartilage of the caprine knee joint post medial meniscectomy and anterior cruciate ligament transection but were detected in regenerated meniscal tissue and synovium3 with associated cartilage protection. This and other studies suggested that the engrafted cells might act to secrete factors that contribute to tissue repair. However, the underlying mechanism of action following intra-articular (IA) MSC injection has not been elucidated.
Content
In an effort to address this lack and define how MSCs act to modulate development or treatment of knee OA, MSCs were retrieved from the joint after IA delivery in a murine collagenase induced osteoarthritis (CIOA) model. The retrieval was performed at times representing early stage or acute OA as well as at a later time with established cartilage degradation in joints. Briefly, CIOA was induced in C57BL/6 mice and syngeneic mouse GFP+ bone marrow derived MSCs (BM-MSCs) were injected into knee joints at days 14 and 56, representing the early acute and established OA joint with cartilage degradation confirmed respectively. For cell retrieval, mice were sacrificed three days following IA-injection and knee joints digested in collagenase type I for six hours before undergoing FACS-coupled cell sorting. Each sample was compared to GFP+ BM-MSCs retrieved from SHAM joints, where the knee joints were injected with saline. Retrieved cells were pooled for analysis of their molecular profile by RNA sequencing. BM-MSCs, licensed in vitro with a single dose of IL-6 (50ng/ml) and a combination of interleukin 6 (IL-6), monocyte chemoattractant protein-1 (MCP-1) and interferon gamma (IFN- γ) each at 50ng/ml were also included as in vitro controls for this analysis. After treatment for 72 hours, these cells were also processed for RNA sequencing. In silico analysis was performed to identify the transcriptome profile of retrieved MSCs in relation to the disease stage. Major canonical pathways and the predicted secretome were also analysed to determine relevant biological processes and functions that can contribute to the ability of MSC to modulate development of OA.
Differentially upregulated genes (DEGs) associated with early retrieval or the initial response of MSCs to joint injury (Day 14) were associated with cell survival, cell proliferation and regulation of apoptosis and autophagy, whereas in late OA-retrieved cells the gene pattern indicated a strong immunomodulatory profile with an increase of genes involved in the complement cascade, secretion of cytokines and M2 macrophage markers. In terms of the predicted secretome, 157 elements were identified as associated with early OA with 278 differentially regulated in response to the established OA environment. The largest cluster for 14D retrieved cells was enriched for metabolism and extracellular matrix organization, whereas immune response and cytokine-cytokine interactions-related genes were dominant in 56D retrieved cells. The Wnt Signalling Pathway was enriched at both time points, with Wnt antagonists SFRP2 and SFRP4 common elements.
In summary, this data characterized the transcriptome profile of therapeutically licensed MSCs exposed to an in vivo OA environment. As a higher number of cells were retrieved from CIOA induced joints compared to SHAM controls, indicating that the inflammatory environment seems to promote cell survival. The surviving cells also responded differently to the disease stage with suggesting that the therapeutic response of MSCs is strongly influenced by the environment to which the cells are delivered. Early stage OA is characterized by an imbalance of the extracellular matrix between anabolic and catabolic responses, therefore it is most likely that the initial response driven by MSCs will be targeting to counteract these processes. In the later disease stage where OA has been established with associated changes to the immune environment, MSCs are driven to express genes mostly involved in immune processes, suggesting a strong immunomodulatory action. However, the molecular response of the surviving MSCs at this time point also suggests potential for cartilage protection and other disease-modifying responses.
References
1. Song et al., J Orthop Translat. 2020 Sep; 24: 121–130 (https://www.ncbi.nlm.nih.gov/labs/pmc/articles/PMC7452318/)
2. Yubo et al., PLoS One. 2017 Apr 27;12(4):e0175449 (https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0175449
3. Murphy et al., Arthritis Rheum 2003 48:3464-74. (https://pubmed.ncbi.nlm.nih.gov/14673997/)
Acknowledgments
Science Foundation Ireland (SFI), the European Regional Development Fund and Abbvie (Grant Number 13/RC/2073)
19.1.3 - Mechano-Induced Cell Communications (Pre-Recorded)
19.1.4 - Extracellular Vesicles for Senescence Targeting in Osteoarthritis and Age-Related Diseases
Abstract
Introduction
Aging is the predominant risk factor for degenerative diseases and conditions that limit health span. Among degenerative rheumatic diseases, the most prevalent is osteoarthritis (OA), which is characterized by the loss of joint function and the dysregulation of several physiological processes. Senescence has been reported to be one of the main drivers of OA pathogenesis, in particular via the release of senescence-associated secretory phenotype (SASP) factors. Understanding senescence occurrence in OA might help to develop novel approaches for treating the disease.
Content
SASP factors are released by chondrocytes, synoviocytes and other joint tissues contributing to disease development and senescent phenotype dissemination. However, the exact mechanism connecting senescence and OA pathology remains unclear. The role of EVs in the propagation of SASP factors and the induction of senescence in the microenvironment and systemically will be discussed in the context of OA.
By contrast to their role in disease dissemination, EVs can have a pro-regenerative role and their rejuvenating function has been demonstrated using EVs from young cells injected into aged animals. This function has been exemplified in vitro using EVs isolated from mesenchymal stromal cells (MSC-EVs). Indeed, infant MSC-EVs or umbilical cord-derived MSC-EVs can rejuvenate elderly or senescing MSCs, as shown by reduced number of senescent cells and ROS accumulation as well as increased self-renewal capacity, telomere length and proliferation rate. Therefore, the possibility that MSC-EVs might exert a senoprotective effect is being investigating. Few data exist on a direct effect of MSC-EVs on OA-associated senescence. A single report on the effect of MSC secretome on the senescent phenotype of OA chondrocytes used the conditioned medium of MSCs.
In the team, we previously demonstrated that MSC-EVs largely mediate the therapeutic effect of parental cells using an in vitro model of OA chondrocytes and in vivo using the collagenase-induced model of OA (CIOA). We will present our recent data on the senoprotective function of MSC-EVs in a new model of etoposide-induced senescence in OA chondrocytes. We used etoposide to induce DNA damage-associated senescence, which was characterized by growth arrest, increase of SA-βGal+ cell number, increase of p15, p21, p27 expression, of nuclear γH2AX foci, of stress fibers and cell surface confirming the induction of main senescence features. The addition of different doses of ASC-EVs at the time of senescence induction prevented the increase of SA-βGal+ cell number and significantly reduced the number of γH2AX+ chondrocytes as well as their nucleus surface. No effect of ASC-EVs was found on CDKI expression whereas the secretion of several SASP factors (IL6, IL8, MMP3, MMP13, HGF, VEGF) was decreased. Interestingly, ASC-EVs slightly up-regulated the anabolic markers of chondrocytes (AGG and type II COLLAGEN) and decreased the expression of catabolic markers (MMP13, AP). The results demonstrate that MSC-EVs exert a senoprotective and chondroprotective effect on OA chondrocytes.
Finally, we will present data on the impact of senescence induction in MSCs and their derived EVs on their therapeutic function in vitro and in vivo models of OA.
References
Boulestreau J, Maumus M, Jorgensen C, Noël D. Extracellular vesicles from mesenchymal stromal cells: therapeutic perspectives for senescence targeting in osteoarthritis. Advanced Drug Delivery Reviews, 2021, 113836.
Acknowledgments
Study was supported by a research grant from FOREUM, Foundation for Research in Rheumatology.