Hypoxia activates the PTHrP – Zfp521 – MEF2C pathway to attenuate hypertrophy in mesenchymal stem cell derived cartilage

Abstract

The treatment of cartilage defects remains an unmet medical need as the current treatment options are currently sub-optimal as the most common treatment options; microfracture and autologous chondrocyte implantation (ACI) result in repair tissue consisting of predominantly fibro-cartilage rather than hyaline cartilage. Due to the ability of mesenchymal stem cells (MSCs) to differentiate along the cartilage lineage, MSCs have been touted as a cellular source to regenerate damaged cartilage. However, a number of prevailing concerns for such a treatment remain. Generally, administration of MSCs into a cartilage defect results in poor regeneration of the damaged cartilage, with most clinical trials reporting that as with microfracture and ACI, the repaired cartilage consists of fibro-cartilage rather than hyaline cartilage. Methods that improve the chondrogenic potential of transplanted MSCs in vivo may be advantageous. In addition, the proclivity of MSC-derived cartilage to undergo hypertrophic differentiation or form bone in vivo also remains a clinical concern. Hypertrophic differentiation of MSC-derived cartilage can be said to closely mimic the endochondral ossification observed in the skeletal development of the long bones of the body. If MSC-derived cartilage was to undergo hypertrophic differentiation in vivo this would result in the failure of the cartilage graft as the newly formed tissue would resemble bone which would lack the ability to absorb and transfer biomechanical loads to the same extent as hyaline cartilage. Physiological hypoxia has previously been shown to improve chondrogenesis of MSC-derived cartilage through upregulation of the chondrogenic transcription factor SRY sex determining region-box 9 (SOX9) and to reduce hypertrophy markers. This study focuses on establishing a mechanism of action by which hypoxia or low oxygen tension can be used to attenuate or limit hypertrophic differentiation of MSC-derived cartilage. Having established a mechanism of action, the subsequent goals of this study were to develop an in vitro culture regime to mimic the beneficial effects of physiological low oxygen tension in a normoxic environment. This was achieved using the pharmacological compound FG-4592, which is currently undergoing clinical trials for the treatment of chronic kidney disease. In conclusion, this study demonstrates that hypoxic differentiation of MSC-derived cartilage has beneficial effects in terms of improved chondrogenesis and attenuated hypertrophy. This study identifies PTHrP, Zfp521 and MEF2C as key factors that regulate hypertrophic differentiation of MSC-derived cartilage in response to physiological hypoxia, genetic activation of the HIF pathway and the hypoxia mimetic compound FG-4592.