Aggrecanolysis and in vitro matrix degradation in the immature bovine meniscus: mechanisms and functional implications

Abstract

Introduction: Little is known about endogenous or cytokine-stimulated aggrecan catabolism in the meniscal fibrocartilage of the knee. The objectives of this study were to characterize the structure, distribution, and processing of aggrecan in menisci from immature bovines, and to identify mechanisms of extracellular matrix degradation that lead to changes in the mechanical properties of meniscal fibrocartilage.

Methods: Aggrecanase activity in the native immature bovine meniscus was examined by immunolocalization of the aggrecan NITEGE neoepitope. To investigate mechanisms of cytokine-induced aggrecan catabolism in this tissue, explants were treated with interleukin-1alpha (IL-1) in the absence or presence of selective or broad spectrum metalloproteinase inhibitors. The sulfated glycosaminoglycan (sGAG) and collagen contents of explants and culture media were quantified by biochemical methods, and aggrecan catabolism was examined by Western analysis of aggrecan fragments. The mechanical properties of explants were determined by dynamic compression and shear tests.

Results: The aggrecanase-generated NITEGE neoepitope was preferentially localized in the middle and outer regions of freshly isolated immature bovine menisci, where sGAG density was lowest and blood vessels were present. In vitro treatment of explants with IL-1 triggered the accumulation of NITEGE in the inner and middle regions. Middle region explants stimulated with IL-1 exhibited substantial decreases in sGAG content, collagen content, and mechanical properties. A broad spectrum metalloproteinase inhibitor significantly reduced sGAG loss, abrogated collagen degradation, and preserved tissue mechanical properties. In contrast, an inhibitor selective for ADAMTS-4 and ADAMTS-5 was least effective at blocking IL-1-induced matrix catabolism and loss of mechanical properties.

Conclusions: Aggrecanase-mediated aggrecanolysis, typical of degenerative articular cartilage, may play a physiologic role in the development of the immature bovine meniscus. IL-1-induced release of sGAG and loss of mechanical properties can be ascribed primarily to the activity of MMPs or aggrecanases other than ADAMTS-4 and ADAMTS-5. These results may have implications for the clinical management of osteoarthritis.

Figure 1

Immature bovine menisci. (a) Coronal sections were harvested from immature bovine menisci for histologic and immunofluorescence analysis and the preliminary tissue culture experiments. (b) Cylindrical explants from the middle region were prepared for the inhibitor study.

Figure 2

Regional variations in proteoglycan content, vasculature, and aggrecanase product abundance (G1-NITEGE) in freshly isolated immature bovine meniscus. (a) Coronal sections of medial meniscus stained for sulfated glycosaminoglycan (red = safranin-O stain; counterstained with fast green), elastin (black = Verhoeff's stain; pink = van Gieson's stain for collagen), or the aggrecan NITEGE neoepitope (green) and collagen type I (red). In immunofluorescent images, the cell nuclei are blue. Inner, middle, and outer refer to the radial position in the coronal plane. Scale bars = 100 μm. (b) The regional variations in aggrecan NITEGE abundance were confirmed by western blot of tissue extracts of both lateral (left lanes) and medial (right lanes) menisci. Equal volumes of tissue extracts (each from about 5 mg wet mass tissue) were loaded in each lane. The migration of globular protein standards are shown at left.

Figure 3

Localization of the NITEGE neoepitope in immature meniscus in the absence and presence of IL-1. Aggrecan NITEGE (green) detected in untreated (top) and IL-1-stimulated (bottom) meniscal fibrocartilage. Cell nuclei are blue. Inner, middle, and outer refer to the radial position in the coronal plane. Scale bar = 100 μm.

Figure 4

Inhibition of IL-1-induced proteoglycan depletion in immature meniscal fibrocartilage. (a) Sulfated glycosaminoglycan (sGAG) contents of middle-zone meniscal fibrocartilage explants. § †, and ‡ indicate P < 0.05 vs. IL-1 for IL-1 + matrix metalloproteinases (MMP) inhibitor, IL-1 + broad-spectrum inhibitor, and untreated, respectively. (b) Aggrecan G1 fragments and decorin core protein in day 0 and day 8 explants. A 5 μg sample of extracted sGAG, pooled from six explants per condition, were loaded in each lane. The doublet migrating at about 65 kDa was confirmed as the G1-NITEGE product (not shown). The migration of globular protein standards are shown at left. Below blots are sGAG contents of the explant extracts from day 8.

Figure 5

Sulfated glycosaminoglycan (sGAG) and aggrecan release from meniscal fibrocartilage to conditioned media. (a) Media from day 12 explants were pooled by time points (days 2 + 4, 6 + 8, and 10 + 12) and assayed for sGAG by the dimethylmethylene blue (DMMB) assay. Data are mean + standard error of the mean with n = 6. § indicates P < 0.05 vs. IL-1. (b) Media were then blotted for aggrecan G1 fragments and NITEGE neoepitope. Equal volumes of media, pooled from six explants per condition, were loaded in each lane. Lane 1 = media pooled from days 2 + 4; Lane 2 = media pooled from days 6 + 8; Lane 3 = media pooled from days 10 + 12. Migration of protein standards are shown at left. In lower panels, arrowheads indicate migration of the 64 kDa molecular weight marker. Note that the early sGAG release data from day 12 explants should not be quantitatively compared with the day 4 residual sGAG contents (Figure 4a) from different explants. MMP = matrix metalloproteinases.

Figure 6

Inhibition of IL-1-induced collagen (a) release to media and (b) loss from immature middle-zone meniscal explants by metalloproteinase inhibitors. Data are mean ± standard error of the mean with n = 6. †, ‡, and # indicate P < 0.05 vs. IL-1 for untreated, IL-1 + matrix metalloproteinase (MMP) inhibitors, and IL-1 + broad-spectrum inhibitors, respectively.

Figure 7

Inhibition of IL-1-induced loss of meniscal explant dynamic (a) compression and (b) shear moduli by metalloproteinase inhibitors. Explants from day 0 and 12 were tested in oscillatory unconfined compression and torsional shear. Data are mean + standard error of the mean, n = 6. ‡ indicate P < 0.05 vs. IL-1. MMP = matrix metalloproteinase.