Agrin: A new player in joint biology and a potent growth factor for cartilage regeneration

Abstract

Purpose: Osteoarthritis is a chronic disabling disease characterized by cartilage breakdown for which there is no cure. Disruption of the gene encoding for the heparan sulphate proteoglycan Agrin results in embryonic skeletal dysplasia suggesting a role for Agrin in cartilage biology and prompting us to study its role in articular cartilage biology and osteoarthritis. The aims of this study are to establish the expression pattern of Agrin in healthy/normal cartilage and compare it to the expression pattern in osteoarthritic or injured cartilage; determine the effects of knockdown and over-expression of Agrin within cartilage in vitro and in vivo and to determine the epistasis of Agrin in articular chondrocytes. Methods: Agrin expression was determined by immunohistochemistry and qPCR. Osteoathritis was induced in 8 week old 129sv mice by destabilisation of the medial meniscus (DMM) and Agrin expression was evaluated by immunofluorescence 8 weeks post-surgery. Paired human samples of preserved cartilage vs severely osteoarthritic cartilage were compared by immunofluorescence. Gain and loss of function experiments were performed using an expression plasmid encoding mammalian Agrin and siRNAs in C28/I2 and bovine chondrocytes in micromass culture. In vivo cartilage formation was assessed using an ectopic implantation model in nude mice; growth-arrested COS7 cells overexpressing Agrin or GFP were combined with bovine chondrocytes (ratio 1:10) and implanted ectopically into nude mice for two weeks. Retrieved implants were characterised by histology and qPCR. Results: Agrin and its known receptors were expressed in healthy adult human articular cartilage and downregulated in human and experimental murine osteoarthritis. Silencing of Agrin by siRNA resulted in reduced GAG production in C28/I2 and in chondrocyte de-differentiation characterised by decreased expression of SOX9, COL2A1 and ACAN mRNA. Overexpression of Agrin in the human chondrocyte cell line C28/I2 and bovine primary articular chondrocytes resulted in enhanced GAG production and SOX9 upregulation. Importantly, in contrast to BMP-2, Agrin over-expression did not induce markers of cartilage hypertrophy including COL10A1 and MMP-13. Delivering Agrin to primary bovine chondrocytes transplanted in the muscle of nude mice resulted in enhanced formation of ectopic cartilage, which did not display signs of hypertrophy, vascular invasion, or endochondral bone formation. Conclusions: Our data show that Agrin is essential for the maintenance of the chondrocytic phenotype and extracellular matrix production whilst exogenous Agrin enhances chondrocyte differentiation and cartilage formation in vitro and in vivo; and therefore may be a valuable chondrogenic molecule in tissue engineering technologies.