The results of this study demonstrate regional variations in the accumulation of aggrecanase-generated aggrecan fragments in the developing bovine meniscus. In articular cartilage, the NITEGE neoepitope is indicative of tissue degeneration and is essentially undetected in immature tissue. In contrast, extensive proteolytic processing of aggrecan, yielding products found in degenerative articular cartilage, appears to be normal in the developing meniscus. NITEGE was abundant in the middle and outer zones of immature meniscal fibrocartilage (as previously reported [13
]) and was steadily released from middle zone explants over 12 days in culture. Interestingly, safranin-O staining is more uniformly distributed and aggrecan gene expression is higher in menisci from mature animals than from immature animals, suggesting that these regional variations in aggrecan density and turnover are dependent on developmental stage [3
]. Although synthesis of proteoglycans can be regulated at the transcriptional (mRNA) level, regional variations in immature meniscus sGAG content may be due in part to regional differences in aggrecanase activity.
Blood vessels were readily detected in the middle and outer regions of the immature meniscus, but absent in the inner region, suggesting a spatial relation between vascular supply and aggrecan destruction in this tissue. It has been suggested that the vasculature and greater oxygen tension of the outer meniscus can suppress cartilage-like matrix formation [41
], and preservation of a chondrogenic phenotype similar to inner region cells may be contingent upon a hypoxic environment [42
]. Further, age-related spatiotemporal changes in meniscal vascular density are consistent with the inversely related changes in sGAG localization, because the early post-natal human meniscus is vascularized throughout while the mature (over 20 years) meniscus is vascularized only in the outer 25 to 33% [43
]. Aggrecanolysis may thus be regulated by access to the meniscal vascular supply or, conversely, serve to regulate vascular persistence in the meniscus. Interestingly, the fat pads of developing mice and the flexor tendons from young and mature bovines were also found to contain NITEGE-bearing aggrecan [44
], suggesting that degradation of aggrecan has physiologic functions in other vascularized tissues as well.
Regional differences in mechanical loading may contribute to the spatial pattern of aggrecanolysis in the meniscus. Finite element models predict substantial regional variations in complex combinations of compressive, tensile, and shear strains during physiologic loading [46
]. Regions of bovine flexor tendon that undergo primarily tensile loading in vivo
synthesized less high molecular weight proteoglycan than explants of compressed tendon [47
]. In addition, cells from the inner and outer regions of the meniscus exhibited distinct biosynthetic responses to biaxial mechanical loading [48
]. It is likely that interactions between soluble and mechanical factors regulate aggrecanolysis in the meniscus, and may contribute to the development of a mechanically appropriate ECM. Extensive aggrecan cleavage may be necessary for the assembly of large tension-bearing collagen fibrils in the middle and outer regions of the meniscus, especially if proper alignment of the collagen fibrils is a cell adhesion-dependent process. Aggrecan inhibits nerve outgrowth partly due to the presence of sGAG, and perineuronal nets are composed of aggrecan [49
]. It is possible that meniscal cells, particularly in the middle and outer regions, would not attach to the ECM or retain a stellate morphology in the presence of full-length aggrecan. There is a direct relation between rounded cell morphology and sGAG abundance in articular cartilage and in the developing meniscus. Maintenance of the stellate morphology may be essential for fibrocartilage-specific mechanotransduction pathways, which in turn regulate ECM remodeling.
IL-1-stimulated menisci stained intensely for NITEGE in the inner and middle regions, where sGAG density was initially highest. These results may actually underestimate the abundance of NITEGE generated in the meniscus because the surface zones of this tissue (removed for the explant experiment) appear to have robust aggrecanase activity (Figure ), consistent with previously reported elevation of MMP-3 expression, aggrecanase activity, and sGAG release from IL-1-treated surface-zone meniscal fibrocartilage from mature bovines [51
]. The sGAG release observed in the current explant study was likely due to the degradation and/or release of aggrecan as well as other proteoglycans. Because the G1-NITEGE fragment of aggrecan, abundant in the immature meniscus, lacks sGAG attachment regions, the contribution of this species to sGAG content and release would be negligible. sGAG was detected in conditioned media throughout the culture period, but high molecular weight aggrecan G1 species were absent from conditioned media collected late in the experiment, suggesting that other proteoglycans contributed to the release of sGAG, particularly at later stages of degradation. IL-1 induced the loss of decorin in this study, and meniscal cells from the immature rabbit express mRNA for another large proteoglycan, versican [52
]. In addition, decorin, biglycan, fibromodulin, and versican have been immunolocalized in fibrocartilage of the intervertebral disc [53
], suggesting that a variety of large and small proteoglycans contribute to the pool of sGAG in the ECM of fibrocartilages. It is important to note that sGAG-bearing aggrecan may be retained within fibrocartilage independent of G1-hyaluronan interactions. Vogel and colleagues detected sGAG-bearing aggrecan lacking the G1 domain in regions of tendon that undergo tensile loading [8
], and we have recently reported that multiple aggrecan G3 fragments accumulate in immature meniscal fibrocartilage [40
]. The G3 domain can bind ECM proteins such as fibulin and tenascin-C [54
], and such interactions could facilitate retention of aggrecan in the meniscus.
The mechanical functions of meniscal fibrocartilage depend on the composition, integrity, and organization of its ECM [56
]. Explanted meniscal fibrocartilage stimulated with IL-1 for 12 days contained 44% and 67% of the sGAG and collagen, respectively, in day 0 tissue. Correspondingly, the depleted tissue retained only 15 to 40% of the shear stiffness and 15 to 22% of the compressive stiffness of the fresh tissue. Explants treated with the MMP inhibitor retained 100% of the initial collagen but only 63% of the initial sGAG. This reduction in sGAG was associated with a 53 to 57% reduction in shear stiffness and a 53 to 61% reduction in compressive stiffness, indicating that despite comprising a quantitatively minor ECM constituent (1 to 3% by dry weight) [57
], proteoglycans contribute substantially to the shear and compression properties of immature bovine meniscal fibrocartilage.
Aggrecanases were mediators of aggrecan proteolysis in the immature meniscus, as evidenced by an abundance of NITEGE in tissue extracts. In contrast, MMPs did not appear to contribute substantially to aggrecanolysis in this system because characteristic MMP-generated aggrecan fragments (identified by G1 bands migrating at about 55 kDa) were not detected in tissue extracts or conditioned media. MMPs did, however, appear to play a strong role in sGAG release from meniscal explants. IL-1-induced loss of sGAG was significantly reduced by treatment with an MMP-selective inhibitor or a broad-spectrum (aggrecanase and MMP) inhibitor but not with an aggrecanase-1,2-selective inhibitor. The selectivities of these inhibitors for aggrecanases other than aggrecanase-1 or -2 (e.g., ADAMTS-1, -8, -9, -15, -16, and -18) are unknown, making it difficult to fully distinguish between the contributions of MMPs and aggrecanases other than ADAMTS-4 or -5 to the release of sGAG in these experiments. However, in a similar model of tissue resorption with age- and species-matched articular cartilage, ADAMTS-4 and -5 mediated 90% of the observed aggrecanase activity and loss of sGAG [58
]. ADAMTS-5 was found to be the dominant aggrecanase in mouse models of arthritis [23
], and a recent report indicates that ADAMTS-8, -15, -16, and -18 are expressed at very low levels in mouse cartilage [59
MMPs may regulate sGAG release by disrupting the aggrecan-hyaluronan aggregate. IL-1-induced depolymerization of hyaluronan by free radicals or hyaluronidases, shown to occur in articular cartilage [60
], may be influenced indirectly by MMP activity (or the inhibitors used here). Link protein, which stabilizes the interaction between aggrecan G1 and hyaluronan, is a substrate for MMP-3 [62
], and CD44, a hyaluronan receptor, is a substrate for MMP-14 [63
]. Release of sGAG from IL-1-stimulated fibrocartilage may be further regulated by MMP-mediated collagen degradation. The organized bundles of collagen fibrils in fibrocartilage may demarcate a metabolic pool of aggrecan for which release is rate-limited by reaction of MMPs with collagen. Akin to the fascicular structures present in tendon and muscle, these structures appear to contain relatively thick fibril bundles. Presumably, MMP-mediated disruption of the fascicular structures would enhance diffusion of collagen-associated aggrecan out of these structures. MMPs thus appear to serve an indirect, but quantitatively significant, role in the release of aggrecan and sGAG from the cytokine-stimulated immature meniscus.