and 2 other(s)
INTRODUCTION: A central pathological feature of osteoarthritis (OA) is the imbalance in articular cartilage extracellular matrix degradation relative to matrix production by chondrocytes. Most evidence indicates that this imbalance is due to increased catabolic processes. However, there is renewed attention to develop strategies that enhance cartilage regenerative capacity based on recent reports of adult chondrocyte pro-genitor cell populations and evidence for endogenous cartilage repair. For example, a recent report identified a population of PRG4+ unipotent chondrocyte progenitor cells (CPCs) present in the superficial layer of articular cartilage in mice (1). Prg4 lineage-tracing experiments showed that PRG4+ superficial zone chondrocytes differentiated into middle-deep zone mature chondrocytes during skeletal development. Thus, OA risk factors that are initiated at young ages, such as obesity, may increase disease risk by impairing CPC function and endogenous repair. Obesity is particularly interesting because progenitor cell differentiation requires metabolic reprogramming to support specialized bioenergetics demands. Therefore, we investigated the effect of obesity and elevated fatty acids on PRG4+ CPC self-renewal, proliferation, and differentiation into chondrocytes. We also tested the role of the mitochondrial deacetylase sirtuin 3 (Sirt3) in CPC proliferation and differentiation into chon-drocytes because it is a central mediator of cellular fatty acid metabolism and anti-oxidant defense and its expression declines with age in cartilage (2, 3). We hypothesized that increased dietary fat and Sirt3 deficiency will reduce CPC proliferation and differentiation. METHODS: All procedures were conducted following an approved IACUC protocol. Bone marrow-derived mesenchymal stem cells (MSCs) were isolated from WT and Sirt3-/- mice and differentiated into chondrocytes using MesenCultTM-ACF chondrogenic medium (STEMCELL) in a 3-D pellet culture model. Additional pellets were cultured with fatty acid supplement (250 µM Oleate+250 µM Palmitate) or BSA control. Pellets were collected between 3-28 days of differentiation and fixed, paraffin-embedded, sectioned, and (immuno-) stained for Safranin O, SIRT3, CPC marker PRG4, and chondrocyte marker COLII (n=3 per group per time point). PRG4 pellet staining was confirmed using anti-GFP staining with MSCs derived from Prg4+/GFPCreERT2 mice. Immuno-fluorescent images were obtained by confocal microscopy, and regional cellular staining intensity was quantified in 8 radial segments and expressed as a relative distance from the pellet center to the edge using Zeiss Zen software. To determine if diet-induced obesity altered cartilage PRG4+ cell populations, knee joint sections from male C57BL/6J mice fed a 60% kcal high fat diet (HF, n=6) or 10% low fat control diet (LF, n=6) for 6 months were immunostained for PRG4. The percentage of PRG+ cells in both lateral and medial tibias were counted under blinded conditions. Results reported as mean ± SEM, with significance evaluated by unpaired Student’s t-test (p<0.05). RESULTS: We confirmed that the chondrogenic pellet culture model recapitulated PRG4+ CPC proliferation and differentiation in vitro. SIRT3 and PRG4 displayed distinct spatial-temporal staining patterns in the chondrogenic pellets. Between 3-10 days, PRG4 expression transitioned from sparse to widespread. Between 14-21 days, 2 regions formed in the pellet--a fibrous outer region with spindle-shaped cells and a uniform central region with round chondrocyte-like cells (Fig. 1). At 21 days, PRG4+ cells were restricted to the fibrous outer region, similar to in vivo superficial-zone cartilage staining patterns. COLII+ cells appeared at day 10 and remained restricted to the central region up to day 28 (Fig 1). SIRT3 was expressed throughout the pellet at day 10 before being primarily restricted to the central region at day 28 (Fig 1). Sirt3-/- chondrogen-ic pellets were smaller, showed less Safranin-O staining, and did not develop an outer PRG4+ circumferential layer (Fig 2). Fatty acid treatment increased perilipin (lipid droplet associated protein) staining; however, FA treatment did not affect the pellet size, Safranin-O staining, or PRG4+ cell numbers or distribution (Fig 2). In mice fed a HF diet for 6 months, the number of PRG+ chondrocytes in the medial (p=0.13) and lateral tibia (p=0.29) were similar to controls (Fig 3). DISCUSSION: We found that a 3-D murine MSC chondrogenic pellet culture model undergoes PRG4+ CPC proliferation and differentiation patterns similar to those observed in vivo, supporting its use as an in vitro model to study CPCs. SIRT3 expression initially tracked PRG4+ cell proliferation throughout the pellet before diverging between days 10-28, suggesting that Sirt3 contributes to specific stages of chondrogenesis. Indeed, Sirt3 deficiency resulted in reduced PRG4+ CPC proliferation and chondrogenic maturation. Our additional hypothesis, that fatty acids would impair CPC function, was not supported as fatty acid treatment did not alter pellet chondrogenesis or alter the number of PRG4+ articular chondrocytes in diet-induced obese mice. We are using Prg4+/GFPCreERT2; Sirt3fl/fl mice to determine if and how Sirt3 expression in PRG4+ CPCs regulates chondrogenesis. SIGNIFICANCE: This work initiates proof-of-concept studies to test the hypothesis that obesity increases OA risk by impairing CPC function, cartilage maturation, and endogenous cartilage repair. If established, these findings could lead to new therapeutic approaches to reduce OA risk using CPC-targeted metabolic therapies. REFERENCES: 1)Kozhemyakina E, et al. 2015.Arthritis & Rheum., 2)Verdin E, et al. 2015 Nat Rev Mol Cell Biol, 3)Fu Y, et al. 2016 Arthritis & Rheum, ACKNOWLEDGEMENTS: Supported by the NIH, the Oklahoma Center for Adult Stem Cell Research a project of TSET, and OCAST.
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