Adenovirus-mediated gene transfer of insulin-like growth factor 1 stimulates proteoglycan synthesis in rabbit joints.

OBJECTIVE: To examine the effect of insulin-like growth factor 1 (IGF-1) on the regulation of cartilage synthesis and other articular events in vivo. METHODS: A first-generation adenoviral vector expressing human IGF-1 (AdIGF-1) from the cytomegalovirus promoter was constructed. Particles of AdIGF-1 (5 x 10(9)) were injected through the patellar tendon into normal rabbit knee joints and rabbit knee joints with antigen-induced arthritis (AIA), with the same dose of a control adenoviral vector injected into the contralateral knees. Lavage fluids were obtained from rabbit knee joints on days 3 and 7 postinjection and used for analysis of IGF-1 expression, white blood cell infiltration, and cartilage breakdown. Cartilage chips from rabbit joints were used for assay of new proteoglycan synthesis, and tissues also were harvested from the dissected knees for histologic study. RESULTS: Intraarticular injection of AdIGF-1 resulted in a mean of 180.6 ng/ml of IGF-1 expression in the lavage fluid from rabbit joints. IGF-1 expression stimulated new proteoglycan synthesis in both naive and AIA rabbit knees, but had no significant chondroprotective or antiinflammatory effects. Histologic analysis showed that elevated levels of IGF-1 expression in both normal and arthritic knees had no adverse pathologic effects on synovium or adjacent muscles. CONCLUSION: Gene transfer of IGF-1 into rabbit knee joints promotes proteoglycan synthesis without significantly affecting inflammation or cartilage breakdown. In addition, no adverse effects following intraarticular IGF-1 gene delivery were observed. Thus, local gene transfer of IGF-1 to joints could serve as a therapeutic strategy to stimulate new matrix synthesis in both rheumatoid arthritis and osteoarthritis.

Nitric oxide inhibits chondrocyte response to IGF-I: inhibition of IGF-IRbeta tyrosine phosphorylation.

Chondrocytes in arthritic cartilage respond poorly to insulin-like growth factor I (IGF-I). Studies with inducible nitric oxide synthase (iNOS) knockout mice suggest that NO is responsible for part of the cartilage insensitivity to IGF-I. These studies characterize the relationship between NO and chondrocyte responses to IGF-I in vitro, and define a mechanism by which NO decreases IGF-I stimulation of chondrocyte proteoglycan synthesis. Lapine cartilage slices, chondrocytes, and cartilage from osteoarthritic (OA) human knees were exposed to NO from the donors S-nitroso-N-acetylpenicillamine (SNAP) or (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1- ium-1, 2-diolate] (DETA NONOate), by transduction with adenoviral transfer of iNOS (Ad-iNOS), or by activation with interleukin-1 (IL-1). NO synthesis was estimated from medium nitrite, and proteoglycan synthesis was measured as incorporation of (35)SO(4). IGF-I receptor phosphorylation was evaluated with Western analysis. SNAP, DETA NONOate, endogenously synthesized NO in Ad-iNOS-transduced cells, or IL-1 activation decreased IGF-I-stimulated proteoglycan synthesis in cartilage and monolayer cultures of chondrocytes. OA cartilage responded poorly to IGF-I; however, the response to IGF-I was restored by culture with N(G)-monomethyl-L-arginine (L-NMA). IGF-I receptor phosphotyrosine was diminished in chondrocytes exposed to NO. These studies show that NO is responsible for part of arthritic cartilage/chondrocyte insensitivity to anabolic actions of IGF-I; inhibition of receptor autophosphorylation is potentially responsible for this effect.

Direct adenoviral gene transfer of viral IL-10 to rabbit knees with experimental arthritis ameliorates disease in both injected and contralateral control knees.

IL-10, a cytokine produced primarily by macrophages, B lymphocytes, and Th2 cells, has both immunostimulatory and immunosuppressive properties. A homologue of IL-10 encoded by EBV, known as viral IL-10 (vIL-10), is also able to suppress the immune response, but may lack some of the immunostimulatory properties of IL-10. To evaluate the potential of vIL-10 to block the progression of rheumatoid arthritis, we have utilized a replication-defective adenovirus vector to deliver the gene encoding vIL-10 to the knee joints of rabbits with Ag-induced arthritis. Intraarticular expression of vIL-10 significantly reduced leukocytosis, cartilage matrix degradation, and levels of endogenous rabbit TNF-alpha, as well as the degree of synovitis, while maintaining high levels of cartilage matrix synthesis. Interestingly, an antiarthritic effect was also observed in opposing contralateral control knee joints that received only a marker gene. An adenoviral vector carrying the enhanced green fluorescent protein marker gene was used to demonstrate that a morphologically similar subset of cells infected in the injected knee joint are able to traffic to the uninjected contralateral knee joint. Our results suggest that direct, local intraarticular delivery of the vIL-10 gene may have polyarticular therapeutic effects.

Nitric oxide in osteoarthritis.

Activated articular chondrocytes produce large amounts of nitric oxide (NO), and there is increasing evidence that this is involved in the etiopathogenesis of osteoarthritis (OA). Because of its short half-life, the biological effects of endogenously produced NO are likely to occur locally within the cartilage. We have observed that inhibitors of NO synthases relieve the inhibition of matrix synthesis that otherwise occurs in response to IL-1. To avoid the use of inhibitors, we have recently transduced chondrocytes with the iNOS (NOS-2) gene and confirmed the ability of the endogenously produced NO to inhibit matrix synthesis. Despite the high levels of NO made by these cells, there was no evidence of apoptosis or other forms of cell death. NO was also shown to inhibit the production of TGF-beta(1)by cells treated with IL-1, as well as to decrease matrix production in response to IGF-1. The hypothesis that NO inhibits matrix production by interfering with important autocrine and paracrine factors should be entertained.

Lessons learned from gene transfer approaches.

Recent technological advances allow the transfer of genes to the synovial lining of joints. As well as opening novel opportunities for therapy, these techniques provide valuable new tools for the study of synovitis and other aspects of the biology of joints in health and disease. This article reviews briefly the results of experiments in which selected genes have been transferred to the knee joints of healthy rabbits and rabbits with antigen-induced arthritis.

The Human Genome Project: implications for the treatment of musculoskeletal disease.

The ultimate goal of the Human Genome Project is the determination of the molecular sequence of the entire human chromosomal complement. Realization of this goal will include characterization of all the genes that cause or predispose to disease, which will most certainly lead to the development of powerful new tools for diagnosis, prevention, and treatment in all medical fields, including orthopaedics. The authors review the fundamentals of human genetics and gene mapping, summarize the progress of the Human Genome Project thus far, and discuss the implications of this research as it relates to the treatment of musculoskeletal diseases.

Constitutive intra-articular expression of human IL-1 beta following gene transfer to rabbit synovium produces all major pathologies of human rheumatoid arthritis.

To investigate the pathophysiologic effects of chronically elevated intra-articular levels of IL-1 beta, we used an ex vivo gene transfer method to deliver and express human IL-1 beta (hIL-1 beta) in the knee joints of rabbits. Expression of hIL-1 beta resulted in a severe, highly aggressive form of arthritis analogous to chronic rheumatoid arthritis in humans. Intra-articular manifestations included intense inflammation, leukocytosis, synovial hypertrophy and hyperplasia, and highly aggressive pannus formation with erosion of the articular cartilage and periarticular bone. Systemic effects were also observed, including diarrhea, fever, weight loss, and an increased erythrocyte sedimentation rate. In addition, the hIL-1 beta was found to induce elevated levels of both rabbit IL-1 beta and TNF-alpha in synovial fluid. Following the loss of hIL-1 beta transgene expression between 14 and 28 days post-transplantation, many of these changes began to normalize. These results suggest that chronically elevated intra-articular levels of IL-1 beta alone are sufficient to produce virtually all the pathologies found in rheumatoid arthritis, and furthermore, demonstrate that gene transfer can be used to investigate the roles of specific gene products in the pathogenesis of arthritis.

A sustained, cytoplasmic transgene expression system delivered by cationic liposomes.

A plasmid containing the reporter gene, chloramphenicol acetyltransferase (CAT), driven by the bacteriophage T7 promoter was co-delivered with purified T7 RNA polymerase by the DC-chol cationic liposomes into human embryonic kidney 293 cells to obtain a transient (2 days) CAT gene expression. To prolong the expression, a T7 autogene which contains the T7 RNA polymerase gene driven by the T7 promoter was included in the transfection complex as a self-amplifying regeneration mechanism for the polymerase. High level CAT gene expression was observed up to 5 days after transfection. This strong and sustained expression system should be useful in gene transfer experiments.

Seasonal patterns of ornithine decarboxylase activity and levels of polyamines in relation to the cytology of germinal cells during spermatogenesis in the sea star, Asterias vulgaris.

The activity of ornithine decarboxylase (ODC) and levels of polyamines were measured in the testes of Asterias vulgaris collected throughout an annual spermatogenic cycle. Samples of the testes were prepared for light and electron microscopy to observe the associated changes in the cytology of germinal cells. The specific activity of ODC increased at the onset of testicular growth, decreased during the coldest period of the winter when testicular growth was minimal, and increased again early in the spring when testes grew maximally. Increased activity of ODC resulted in increased levels of polyamines and was correlated with either mitotic or meiotic germinal cell divisions, or both. Spermine levels were always greater than putrescine, followed by spermidine. Highest levels of polyamine synthesis coincided with the onset of spermatogonial proliferation during the fall and with the period of meiotic differentiation and spermiogenesis in the spring. Mid-summer (July) testes were small (0.3-0.5 gonad index (GI)) and contained amitotic spermatogonia arrested in G(1) of the cell cycle. Mitotic and pre-meiotic testes (October/November) increased slightly in size (0.3-1.4 GI) and contained actively dividing spermatogonia, most of which differentiated into primary spermatocytes. Testes from February and March were large (1-6.75 GI), but the proliferative status of their spermatogonia and primary spermatocytes varied. Spermatogonia and primary spermatocytes from early February testes were not dividing. In testes obtained in March, both spermatogonial mitosis and meiosis of spermatocytes resumed, coincident with increased field water temperatures.