Human meniscus cells express hypoxia inducible factor-1alpha and increased SOX9 in response to low oxygen tension in cell aggregate culture

In previous work we demonstrated that the matrix-forming phenotype of cultured human cells from whole meniscus was enhanced by hypoxia (5% oxygen). Because the meniscus contains an inner region that is devoid of vasculature and an outer vascular region, here we investigate, by gene expression analysis, the separate responses of cells isolated from the inner and outer meniscus to lowered oxygen, and compared it with the response of articular chondrocytes. In aggregate culture of outer meniscus cells, hypoxia (5% oxygen) increased the expression of type II collagen and SOX9 (Sry-related HMG box-9), and decreased the expression of type I collagen. In contrast, with inner meniscus cells, there was no increase in SOX9, but type II collagen and type I collagen increased. The articular chondrocytes exhibited little response to 5% oxygen in aggregate culture, with no significant differences in the expression of these matrix genes and SOX9. In both aggregate cultures of outer and inner meniscus cells, but not in chondrocytes, there was increased expression of collagen prolyl 4-hydroxylase (P4H)α(I) in response to 5% oxygen, and this hypoxia-induced expression of P4Hα(I) was blocked in monolayer cultures of meniscus cells by the hypoxia-inducible factor (HIF)-1α inhibitor (YC-1). In fresh tissue from the outer and inner meniscus, the levels of expression of the HIF-1α gene and downstream target genes (namely, those encoding P4Hα(I) and HIF prolyl 4-hydroxylase) were significantly higher in the inner meniscus than in the outer meniscus. Thus, this study revealed that inner meniscus cells were less responsive to 5% oxygen tension than were outer meniscus cells, and they were both more sensitive than articular chondrocytes from a similar joint. These results suggest that the vasculature and greater oxygen tension in the outer meniscus may help to suppress cartilage-like matrix formation. Introduction The meniscus serves as a critical fibrocartilaginous tissue in the biomechanics of the knee joint, and it plays an important role in load distribution and joint stability [1,2]. Its biomechanical importance is further highlighted by the high incidence of osteoarthritis after menisectomy [3-8]. The function of the meniscus is reflected in its cellular and biochemical composition, which ensures that shear, tensile and compressive forces are appropriately distributed in the knee joint [9]. The meniscus exhibits regional and zonal variations in its cellular composition [9-13], reparative capacity [14,15] and microstructure [16,17]. The cells of the outer one-third are fibroblast-like, with extensive cellular processes that may stain positively for CD34 and are within a dense connective tissue, which is composed predominantly of type I collagen fibre bundles aligned in the circumferential direction of the tissue, along with smaller amounts of proteoglycans and minor collagens including types III and V [16,18-21]. In contrast, cells from the middle and DMEM = Dulbecco's modified Eagle's medium; FCS = foetal calf serum; HIF = hypoxia inducible factor; P4H = prolyl 4-hydroxylase; PHD = HIF prolyl-hydroxylase; SOX9 = Sry-related HMG box-9. Arthritis Research & Therapy Vol 9 No 4 Adesida et al.