Dual transduction of insulin‐like growth factor‐I and interleukin‐l receptor antagonist protein controls cartilage degradation in an osteoarthritic culture model

Abstract

This study evaluated the potential of gene induced synoviocyte expression of a combination of insulin-like growth factor-I (AdIGF-I) and interleukin-1 receptor antagonist protein (AdIL-1Ra) to control articular cartilage degradation in vitro. Cartilage explants and synovial membrane were harvested from young mature horses. Synovial monolayers were established and either (1) maintained as untransduced controls; (2) transduced with AdIGF-I at 200 MOI in 500 pl serum-free medium; (3) transduced with AdIL-1Ra at 100 MOI; or (4) transduced with a combination of AdIGF-I (200 MOI) and AdIL-1Ra (100 MOI). Following transduction, cartilage explants were exposed to the synovial monolayer medium using co-culture inserts. Cultures were maintained for 6days in either serum-free medium or medium containing 10 ng/ml recombinant human interleukin-1 p. At termination, synovial cell RNA was isolated for real-time PCR analysis, and cartilage explants were collected for H&E and toluidine blue staining, immunohistochemistry for type I1 collagen and IGF-I, in situ localization of IGF-I and type I1 collagen gene expression, and biochemical assays. Synovial monolayers were readily transduced with both AdIGF-I and AdIL-1 Ra. IGF-I and IL-1 Ra protein were secreted at beneficial levels throughout the experiment, having peak concentrations of 94.6 nglml and 33.0 ng/ml, respectively. Transduction with IGF-I promoted cartilage production of proteoglycan and type I1 collagen, suggesting a beneficial role for healing injured cartilage. Transduction with IL-1Ra decreased the synovial expression of IL-la and IL-1 p and matrix metalloproteinases, indicating a mechanism for prevention of matrix degradation. The beneficial effects of the combination of anabolic growth factors and catabolic blockers were evident in improved preservation of proteoglycan content of cartilage explants exposed to the depleting effects of IL-I . These results show that gene therapy combining anabolic growth factors to stimulate matrix synthesis and catabolic blockers to prevent matrix degradation by IL-1, protects and causes partial restoration of cartilage matrix, and suggest a potential benefit of combination gene therapy for cartilage healing.

Benefits of Recombinant Adeno-Associated Virus (rAAV)-Mediated Insulinlike Growth Factor I (IGF-I) Overexpression for the Long-Term Reconstruction of Human Osteoarthritic Cartilage by Modulation of the IGF-I Axis

“…Among the candidates tested for their reparative properties, IGF-I was described for its ability to modulate cell proliferation and extracellular matrix synthesis in horse and rabbit experimental models of cytokine-induced cartilage matrix degradation in situ (13,14,22). Yet, to the best of our knowledge, the effects of IGF-I gene transfer upon the longterm remodeling of human OA cartilage have not been evaluated to date.…”

“…Current approaches that aim at re-equilibrating the metabolic balance in OA cartilage are on the basis of the transfer of sequences coding for agents that either counteract the processes of matrix degradation or enhance the synthesis of matrix components. Protective effects against cartilage breakdown have been documented in experimental models of OA using sequences coding for inhibitors of inflammatory pathways (an IL-1 receptor antagonist [IL-1Ra], soluble TNF receptor [sTNFR], tissue inhibitor of metalloproteinases [TIMP-1]) (6)(7)(8)(9)(10)(11)(12)(13)(14) or factors such as IL-10 (12), heat shock protein 70 (15), glutamine:fructose-6-phosphate amidotransferase (16), thrombospontin-1 (17), kallistatin (18) and inhibitors of nuclear factor κB (19). Even though application of these stimuli was capable of containing cartilage degradation, it was not sufficient to compensate for the loss of matrix elements and cells and to reestablish an original cartilage surface.…”

“…Growth, transcription and enzymatic factors are potent candidates to achieve these goals because of their anabolic or mitogenic properties such as fibroblast growth factor 2 (FGF-2) (20,21), bone morphogenetic proteins 2 and 4 (BMP-2 and BMP-4) (22,23), transforming growth factor β (TGF-β) (22,24,25), transcription factor sex-determining region Y-type high mobility group box 9 (SOX9) (20,26,27), glucuronosyltransferase-I (28), bcl-2 (29) and telomerase (30). A critical agent that may have the strongest value to readjust the disturbed homeostasis in OA cartilage is insulinlike growth factor (IGF)-I, since it has the ability to influence concomitantly metabolic and proliferative processes, affording protection against extracellular matrix degradation in horse and rabbit articular cartilage explant cultures experimentally treated with proinflammatory cytokines (13,14,22).…”

“…In contrast with vectors derived from adenoviruses (6,10,13,14,(16)(17)(18)(19)21,22,25) and retroviruses (7,11,12,23,26,30) or with nonviral compounds (8,15,28,29), systems based on the replication-defective, nonpathogenic human adeno-associated virus (AAV) may provide better tools for OA, since recombinant AAV (rAAV) can deliver genes in nondividing cells such as chondrocytes both in vitro and in situ in their dense extracellular matrix at high efficiencies and for extended periods of time (20,24,27,31). Also, removal of the viral protein coding sequences in rAAV make them less immunogenic than adenoviral vectors characterized by shortterm transgene expression levels (32).…”

Cartilage tissue engineering for degenerative joint disease☆

“…Dual expression of IGF-I and interleukin-1 receptor antagonist (IL-1Ra) were studied in the horse OA model [160]. Cartilage explants were exposed to the milieu of monolayer synovial membrane-derived cells expressing IGF-1 and IL-1Ra.…”