Approaches to enhancing the retroviral transduction of human synoviocytes.

This report concerns a clinical trial for rheumatoid arthritis (RA), approved by the US National Institutes of Health and the Food and Drug Administration. An amphotropic retrovirus (MFG-IRAP) was used ex vivo to transfer a cDNA encoding human interleukin-1 receptor antagonist (IL-1Ra) to synovium. The protocol required the transduced cells to secrete at least 30 ng IL-1Ra/10(6) cells per 48 h before reimplantation. Here we have evaluated various protocols for their efficiency in transducing cultures of human rheumatoid synoviocytes. The most reliably efficient methods used high titer retrovirus (approximately 10(8) infectious particles/ml). Transduction efficiency was increased further by exposing the cells to virus under flow-through conditions. The use of dioctadecylamidoglycylspermine (DOGS) as a polycation instead of Polybrene (hexadimethrine bromide) provided an additional small increment in efficiency. Under normal conditions of static transduction, standard titer, clinical grade retrovirus (approximately 5 x 10(5) infectious particles/ml) failed to achieve the expression levels required by the clinical trial. However, the shortfall could be remedied by increasing the time of transduction under static conditions, transducing under flow-through conditions, or transducing during centrifugation.

Increased matrix synthesis following adenoviral transfer of a transforming growth factor beta1 gene into articular chondrocytes.

Monolayer cultures of lapine articular chondrocytes were transduced with first-generation adenoviral vectors carrying lacZ or transforming growth factor beta1 genes under the transcriptional control of the human cytomegalovirus early promoter. High concentrations of transforming growth factor beta1 were produced by chondrocytes following transfer of the transforming growth factor beta1 gene but not the lacZ gene. Transduced chondrocytes responded to the elevated endogenous production of transforming growth factor beta1 by increasing their synthesis of proteoglycan, collagen, and noncollagenous proteins in a dose-dependent fashion. The increases in collagen synthesis were not accompanied by alterations in the collagen phenotype; type-II collagen remained the predominant collagen. Transforming growth factor beta1 could not, however, rescue the collagen phenotype of cells that had undergone phenotypic modulation as a result of serial passaging. These data demonstrate that chondrocytes can be genetically manipulated to produce and respond to the potentially therapeutic cytokine transforming growth factor beta1. This technology has a number of experimental and therapeutic applications, including those related to the study and treatment of arthritis and cartilage repair.

Genetic enhancement of matrix synthesis by articular chondrocytes: comparison of different growth factor genes in the presence and absence of interleukin-1.

OBJECTIVE: To determine whether articular chondrocytes express growth factor genes delivered by adenoviral vectors and whether expression of these genes influences matrix synthesis in the presence and absence of interleukin-1 (IL-1). METHODS: Monolayer cultures of rabbit articular chondrocytes were infected with recombinant adenovirus carrying genes encoding the following growth factors: insulin-like growth factor 1 (IGF-1), transforming growth factor beta1 (TGFbeta1), and bone morphogenetic protein 2 (BMP-2). As a control, cells were transduced with the lac Z gene. Cultures were also treated with each growth factor supplied as a protein. Levels of gene expression were noted, and the synthesis of proteoglycan, collagen, and noncollagenous proteins was measured by radiolabeling. Collagen was typed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The effects of growth factor gene transfer on proteoglycan synthesis in the presence of IL-1 were also measured. RESULTS: The expression of all transgenes was high following adenoviral transduction. Proteoglycan synthesis was stimulated approximately 8-fold by the BMP-2 gene and 2-3-fold by the IGF-1 gene. The effects of BMP-2 and IGF-1 genes were additive upon cotransduction. Synthesis of collagen and noncollagenous proteins, in contrast, was most strongly stimulated by the IGF-1 gene. In each case, collagen typing confirmed the synthesis of type II collagen. IL-1 suppressed proteoglycan synthesis by 50-60%. IGF-1 and TGFbeta genes restored proteoglycan synthesis to control levels in the presence of IL-1. The BMP-2 gene, in contrast, elevated proteoglycan synthesis beyond control levels in the presence of IL-1. CONCLUSION: Transfer of growth factor genes to articular chondrocytes can greatly increase matrix synthesis in vitro, even in the presence of the inflammatory cytokine IL-1. This result encourages the further development of gene therapy for the repair of damaged cartilage.

Does nitric oxide help explain the differential healing capacity of the anterior cruciate, posterior cruciate, and medial collateral ligaments?

This study compared the ability of rabbit medial collateral ligament, posterior cruciate ligament, and anterior cruciate ligament tissue to synthesize nitric oxide, and determined its effects on matrix synthesis, an important component of ligament repair. It is not known whether ligament cells can produce nitric oxide and, if so, whether it influences healing of ligament injuries. The anterior cruciate and posterior cruciate ligament tissue produced large amounts of nitric oxide in response to the inflammatory cytokine interleukin-1. Medial collateral ligament, in contrast, produced only modest amounts of nitric oxide. Furthermore, anterior cruciate ligament and, to some degree, posterior cruciate ligament synthesized nitric oxide spontaneously in culture, whereas medial collateral ligament never did so. When nitric oxide was supplied to these tissues, it strongly inhibited collagen synthesis by the two cruciate ligaments, but had little effect on collagen synthesis by the medial collateral ligament. Endogenously synthesized nitric oxide was also able to inhibit collagen synthesis as well as proteoglycan synthesis by the two cruciate ligaments, but had little effect on matrix synthesis by the medial collateral ligament. We propose a novel hypothesis, based on nitric oxide production and matrix synthesis, that may help explain why the two cruciate ligaments have such limited healing capacity compared with the medial collateral ligament.

[In vitro transduction of human osteoblast cell populations with retroviral vectors]. / In vitro Transduktion humaner osteoblastärer Zellpopulationen mit retroviralen Vektoren.

OBJECTIVES: The involvement of cytokines in degeneration and inflammation of human tissue is well established. Interleukin-1 (IL-1) is a major agent in the pathophysiology of periarticular bone resorption in rheumatoid arthritis and in osteoporosis. Because the use of recombinant cytokines and growth factors is limited due to their short half lives, techniques are needed to get a permanent release of these therapeutic proteins. The rational of this study was to show that retroviral transduction of human osteoblastic cells is possible in vitro using the marker gene LacZ and the potentially therapeutic gene encoding for human interleukin-1 receptor antagonist protein (IL-1Ra). Different transduction techniques were combined to improve the rate of transduction in vitro. METHODS: Osteoblastic cells were isolated from human spongious bone and cultured in vitro. The beta-galactosidase (LacZ) gene and the cDNA of IL-1Ra were introduced into the isolated cells by retrovirus mediated gene transfer. LacZ activity was determined by Xgal staining, IL-1Ra was measured quantitatively by ELISA. RESULTS: The transfer of retroviral IL-1Ra led to IL-1Ra expression of 8614 to 10,089 pg IRAP/50,000 cells/48 h. By combining different techniques to improve transduction, the X-gal staining established a rate of transduction of 60%. CONCLUSION: Our results demonstrate that retroviral transduction of human osteobalstic cells is possible in vitro, and leads to high levels of the synthesized transgene product. The rate of retroviral transduction can be accelerated in vitro.

Early expression of marker genes in the rabbit medial collateral and anterior cruciate ligaments: the use of different viral vectors and the effects of injury.

Gene therapy is a technique that may offer advantages over current methods of cytokine delivery to ligaments. To determine if implanted genes could be expressed in normal and injured knee ligaments, the medial collateral ligament and anterior cruciate ligament were studied in 18 rabbits. A retroviral ex vivo technique using allograft medial collateral ligament and anterior cruciate ligament fibroblasts and an adenoviral in vivo technique were compared as methods for delivering the LacZ marker gene to knee ligaments. Bilateral knee surgeries were performed, and the rabbits were equally divided into three groups. Group 1 received the retrovirus and the medial collateral ligament was ruptured, Group 2 received the adenovirus and the medial collateral ligament was ruptured, and Group 3 received the adenovirus and the medial collateral ligament was not injured. The anterior cruciate ligament was not injured in any group. The medial collateral and anterior cruciate ligaments of the right knees received 10(6) allografted, transduced ligament fibroblasts or 10(9) adenovirus particles, whereas the ligaments of the left knee received a similar volume of saline solution only. Equal numbers of rabbits were killed at 10 days, 3 weeks, and 6 weeks following the procedure. Ligament samples were stained with X-gal to detect the expression of the LacZ gene product, beta-galactosidase. LacZ gene expression was evident in ruptured and uninjured medial collateral ligaments as well as in the anterior cruciate ligament. The expression lasted between 10 days and 3 weeks in the medial collateral and anterior cruciate ligaments with use of the retrovirus and between 3 and 6 weeks in the medial collateral ligament and at least 6 weeks in the anterior cruciate ligament with the adenovirus. The length of gene expression in the ruptured and uninjured medial collateral ligaments did not differ. These preliminary studies indicate that gene transfer to normal and injured knee ligaments is possible.

Inhibition of transforming growth factor beta production by nitric oxide-treated chondrocytes: implications for matrix synthesis.

OBJECTIVE: Nitric oxide (NO) is generated copiously by articular chondrocytes activated by interleukin-1beta (IL-1beta). If NO production is blocked, much of the IL-1beta inhibition of proteoglycan synthesis is prevented. We tested the hypothesis that this inhibitory effect of NO on proteoglycan synthesis is secondary to changes in chondrocyte transforming growth factor beta (TGFbeta). METHODS: Monolayer, primary cultures of lapine articular chondrocytes and cartilage slices were studied. NO production was determined as nitrite accumulation in the medium. TGFbeta bioactivity in chondrocyte- and cartilage-conditioned medium (CM) was measured with the mink lung epithelial cell bioassay. Proteoglycan synthesis was measured as the incorporation of 35S-sodium sulfate into macromolecules separated from unincorporated label by gel filtration on PD-10 columns. RESULTS: IL-1beta increased active TGFbeta in chondrocyte CM by 12 hours; by 24 hours, significant increases in both active and latent TGFbeta were detectable. NG-monomethyl-L-arginine (L-NMA) potentiated the increase in total TGFbeta without affecting the early TGFbeta activation. IL-1beta stimulated a NO-independent, transient increase in TGFbeta3 at 24 hours; however, TGFbeta1 was not changed. When NO synthesis was inhibited with L-NMA, IL-1beta increased CM concentrations of TGFbeta1 from 24-72 hours of culture. L-arginine (10 mM) reversed the inhibitory effect of L-NMA on NO production and blocked the increases in TGFbeta1. Anti-TGFbeta1 antibody prevented the restoration of proteoglycan synthesis by chondrocytes exposed to IL-1beta + L-NMA, confirming that NO inhibition of TGFbeta1 in IL-1beta-treated chondrocytes effected, in part, the decreased proteoglycan synthesis. Furthermore, the increase in TGFbeta and proteoglycan synthesis seen with L-NMA was reversed by the NO donor S-nitroso-N-acetylpenicillamide. Similar results were seen with cartilage slices in organ culture. The autocrine increase in CM TGFbeta1 levels following prior exposure to TGFbeta1 was also blocked by NO. CONCLUSION: NO can modulate proteoglycan synthesis indirectly by decreasing the production of TGFbeta1 by chondrocytes exposed to IL-1beta. It prevents autocrine-stimulated increases in TGFbeta1, thus potentially diminishing the anabolic effects of this cytokine in chondrocytes.

Generation of nitric oxide by lapine meniscal cells and its effect on matrix metabolism: stimulation of collagen production by arginine.

Slices of lapine meniscus produced large amounts of nitric oxide after stimulation with interleukin-1, tumor necrosis factor alpha, or a mixture of lapine synovial cytokines known as chondrocyte-activating factors. Monolayer cultures of meniscal cells produced from the proteolysis of meniscal tissue contained a mixed population of chondrocytic and fibroblastic cells. These cultures also produced large amounts of nitric oxide in response to cytokines. Monolayer cultures of meniscal cells produced by the explant method, in contrast, were uniformly fibroblastic and did not produce nitric oxide in response to cytokines. We conclude that menisci contain two populations of cells, one fibroblastic and the other chondrocytic. The chondrocytic cells are responsible for generating most of the nitric oxide in response to cytokines. Endogenously generated nitric oxide suppressed the synthesis of collagen and proteoglycan by menisci but protected proteoglycan from the catabolic effects of interleukin-1. The inhibitory effect of nitric oxide on collagen synthesis occurred without greatly altering the abundance of mRNAs encoding the various collagen alpha chains. During further investigation, arginine was unexpectedly found to stimulate the synthesis of collagen and, to a lesser degree, of noncollagenous proteins but not of proteoglycans. Fragments of meniscus, but not meniscal cells in monolayer culture, increased their production of matrix metalloproteinases, lactate, and, especially, prostaglandin E2 in response to interleukin-1. Inhibition of nitric oxide production with NG-monomethyl-L-arginine enhanced production of matrix metalloproteinases but had little effect on the synthesis of lactate or prostaglandin E2.

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.

The effects of age on rabbit MCL fibroblast matrix synthesis in response to TGF-beta 1 or EGF.

In this study, we examined the effects of age on collagen and total protein synthesis by ligament fibroblasts in response to growth factors. Three different doses of transforming growth factor-beta 1 (TGF-beta 1) or epidermal growth factor (EGF) were individually added to in vitro fibroblast cultures from the medial collateral ligament (MCL) of skeletally immature (age 3 months), mature (age 12 months) and senescent (age 48-51 months) rabbits. Analysis of the effects of age revealed that fibroblasts from senescent rabbits produced significantly less collagen in response to TGF-beta 1 or EGF stimulation when compared to fibroblasts from immature rabbits. Furthermore, increased age was found to result in significant reductions in the baseline levels of collagen synthesis but not total protein synthesis. Additionally, collagen and total protein synthesis by MCL fibroblasts were significantly affected by the TFG-beta 1 dose, but not by the EGF dose. When fibroblasts were normalized to their own controls, the increase in collagen and total protein synthesis due to TGF-beta 1 and EGF for the senescent group were found to be greater than those for the skeletally immature rabbits at all doses. This demonstrates that MCL fibroblasts from senescent rabbits are responsive to growth factors.

In vivo suppression of early experimental osteoarthritis by interleukin-1 receptor antagonist using gene therapy.

OBJECTIVE: This study explored the therapeutic effect of interleukin-1 receptor antagonist (IL-1Ra), administered by gene transfer, on the progression of osteoarthritic (OA) lesions in an experimental dog model. METHODS: Seventeen mature mongrel dogs were divided into 3 groups. Group 1 (n = 7) had an anterior cruciate ligament (ACL) section of the right knee through a stab wound incision. Groups 2 and 3 (n = 5 per group), had an ACL section of the right knee and partial synovectomy of the left knee. Each dog's synovium was subjected to enzymatic digestion, and the synovial fibroblasts were propagated in monolayer culture. Synovial cells from each dog were transduced in vitro using the retrovirus MFG with either the Escherichia coli beta-galactosidase (lac Z) gene (group 2) or the human IL-1Ra gene (group 3). Two days after surgery, the dogs received intraarticular injections as follows: group 1 phosphate buffered saline (PBS) (2 ml); group 2 autologous cells (60 x 10(6) cells/2 ml of PBS) transduced with the lac Z gene; group 3 autologous cells transduced with the IL-1Ra gene. Synovial fluid was aspirated at 2 weeks and 4 weeks. All dogs were euthanized at 4 weeks postsurgery. The right knees were dissected, and lesions were scored for macroscopic and microscopic changes. Synovial explants were dissected and representative specimens were used for histology or were cultured for 48 hours. The levels of IL-1Ra in synovial fluid and synovial explant conditioned medium were measured by specific enzyme-linked immunosorbent assay. RESULTS: The level of IL-1Ra in synovial fluid of group 3 was 202.8 +/- 131.5 ng/ml (mean +/- SEM) at 2 weeks and 2.8 +/- 2.2 ng/ml at 4 weeks after surgery. Membrane explants isolated from dogs that received synovial cells transduced with the IL-1Ra gene (group 3) actively produced IL-1Ra (4.0 +/- 2.0 ng/gm of tissue wet weight). The severity of OA cartilage lesions was similar in groups 1 and 2. In contrast, group 3 dogs had a marked reduction in macroscopic lesion severity on the tibial plateaus (P < 0.01 for grade; P < 0.04 for size) and femoral condyles. Moreover, the histologic lesion severity was decreased on both plateaus (P < 0.06) and condyles. CONCLUSION: This study showed that a local increase in IL-1Ra production in OA knee joints by intraarticular injection of transduced synovial cells can reduce the progression of experimentally induced lesions.

Toward a biochemical understanding of human intervertebral disc degeneration and herniation. Contributions of nitric oxide, interleukins, prostaglandin E2, and matrix metalloproteinases.

STUDY DESIGN: Normal and herniated human intervertebral disc specimens were cultured to study the effects of interleukin-1 beta on the production of nitric oxide, interleukin-6, prostaglandin E2, and matrix metalloproteinases. The effects of endogenously produced nitric oxide on the synthesis of other mediators also were studied. OBJECTIVES: To test the hypothesis that the cells of the intervertebral disc are metabolically active and are capable of responding to biochemical stimuli such as interleukin-1 beta in a manner that could engender degenerative changes. As part of this study, the authors also investigated some of the possible autocrine regulatory mechanisms that may operate during the biochemical responses of disc cells. SUMMARY OF BACKGROUND DATA: The authors previously showed, for the first time, that herniated cervical and lumbar disc specimens spontaneously produce increased amounts of nitric oxide, interleukin-6, prostaglandin E2, and certain matrix metalloproteinases. These results suggest that these biochemical agents are in some manner involved with degenerative processes in the intervertebral disc. This novel hypothesis merits further evaluation; the current communication reports the results of experiments designed to do so. METHODS: Fourteen normal, nondegenerated discs (control group) were obtained from seven patients undergoing anterior spinal surgery for trauma or lumbar scoliosis. Thirty-six herniated discs (18 lumbar and 18 cervical) were obtained from 30 patients undergoing surgery for persistent radiculopathy. The specimens were placed into tissue culture and incubated for 72 hours in the presence or absence of interleukin-1 beta and NG-monomethyl-L-arginine, and inhibitor of nitric oxide synthases, and the media were subsequently collected for biochemical analysis. Biochemical assays for matrix metalloproteinases, nitric oxide, interleukin-6, and prostaglandin E2 were performed. RESULTS: Normal, control disc specimens significantly increased their production of matrix metalloproteinases, nitric oxide, interleukin-6, and prostaglandin E2 in response to interleukin-1 beta. Herniated lumbar and cervical discs, which were spontaneously releasing increased levels of these biochemical agents, further increased their production of nitric oxide, interleukin-6, and prostaglandin E2 in response to interleukin-1 beta. Blocking the biosynthesis of nitric oxide in interleukin-1 beta-stimulated disc cells provoked a large increase in the production of interleukin-6. CONCLUSIONS: Cells of the intervertebral discs are biologically responsive and increase their production of matrix metalloproteinases, nitric oxide, interleukin-6, and prostaglandin E2 when stimulated by interleukin-1 beta. The effect is more dramatic in normal, nondegenerated discs where spontaneous synthesis of these mediators is low. Nevertheless, cells of the herniated degenerated discs where spontaneous production was high were still capable of further increasing their synthesis of several of these biochemical agents in response to interleukin-1 beta. Endogenously produced nitric oxide appears to have a strong inhibitory effect on the production of interleukin-6, which suggests that autocrine mechanisms play an important role in the regulation of disc cell metabolism.

Nitric oxide inhibits the synthesis of type-II collagen without altering Col2A1 mRNA abundance: prolyl hydroxylase as a possible target.

The addition of human recombinant interleukin-1beta (IL-1beta) to cultures of lapine articular chondrocytes provoked the synthesis of large amounts of NO and reduced the production of type-II collagen. NG-Monomethyl-l-arginine (L-NMA), an inhibitor of NO synthase, strongly suppressed the production of NO and partially relieved the inhibition of collagen synthesis in response to IL-1beta. The NO donor S-nitrosoacetylpenicillamine (SNAP), on the other hand, inhibited collagen production. IL-1 lowered the abundance of Col2A1 mRNA in an NO-independent manner. Collectively, these data indicate that IL-1 suppresses collagen synthesis at two levels: a pretranslational level which is NO-independent, and a translational or post-translational level which is NO-mediated. These effects are presumably specific as L-NMA and SNAP had no effect on total protein synthesis or on the distribution of newly synthesized proteins between the cellular and extracellular compartments. Prolyl hydroxylase is an important enzyme in the post-translational processing of collagen, and its regulation and cofactor requirements suggest possible sensitivity to NO. Extracts of cells treated with IL-1 or SNAP had lower prolyl hydroxylase activity, and L-NMA was partially able to reverse the effects of IL-1. These data suggest that prolyl hydroxylase might indeed be a target for NO. Because underhydroxylated collagen monomers fail to anneal into stable triple helices, they are degraded intracellularly. Inhibition of prolyl hydroxylase by NO might thus account for the suppressive effect of this radical on collagen synthesis.

Effect of growth factors on matrix synthesis by ligament fibroblasts.

Although it has been reported that several growth factors modulate soft-tissue healing, the specific effects of growth factors on protein synthesis during ligament healing have not been widely investigated. In this study, we examined the effects of basic and acidic fibroblast growth factors, transforming growth factor beta 1, and epidermal growth factor on collagen and noncollagenous protein synthesis by cultured fibroblasts from medial collateral ligament and anterior cruciate ligament in vitro. Uptake of tritiated proline was used to measure synthesis of collagen and noncollagenous protein, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to analyze the type of collagens synthesized. Our data showed that transforming growth factor beta 1 increased both collagen and noncollagenous protein synthesis by medial collateral and anterior cruciate ligament fibroblasts on a dose-dependent basis. Collagen synthesis by cultured fibroblasts from the medial collateral and anterior cruciate ligaments was increased by treatment with transforming growth factor beta 1 by as much as approximately 1.5 times that of untreated controls. Although the response to transforming growth factor beta 1 by anterior cruciate ligament fibroblasts was equal to that by medial collateral ligament fibroblasts, the amounts of matrix proteins synthesized by anterior cruciate ligament fibroblasts were approximately half of that by medial collateral ligament fibroblasts. The increase was mostly in type-I collagen. Treatment of anterior cruciate ligament fibroblasts with epidermal growth factor increased collagen synthesis by approximately 25% but had little effect on medial collateral ligament fibroblasts. Neither basic nor acidic fibroblast growth factor increased either collagen or noncollagenous protein synthesis. These findings suggest that topical application of transforming growth factor beta 1, alone or in combination with epidermal growth factor, may have the potential to strengthen the ligament by increasing matrix synthesis during its remodeling and healing processes.

Effect of growth factors on the proliferation of ligament fibroblasts from skeletally mature rabbits.

Growth factors have been shown to stimulate fibroblast proliferation during wound and ligament healing. In this study, we analyzed individual effects of eight growth factors in vitro on the proliferation of fibroblasts from the medial collateral (MCL) and anterior cruciate (ACL) ligaments of skeletally mature rabbits. We compared the proliferative response of growth factor-treated and nontreated fibroblasts of both ligaments. The growth-factor treated fibroblasts of the MCL and ACL were also compared. We found that the fibroblasts exposed to epidermal growth factor, basic fibroblast growth factor and platelet-derived growth factor-BB proliferated significantly more than untreated fibroblasts. Acidic fibroblast growth factor at a dose of 1.0 ng/ml caused significant increases in fibroblast proliferation only in the MCL. Transforming growth factor-beta 1, insulin-like growth factor-1, platelet-derived growth factor-AA, and interleukin-1 alpha did not significantly stimulate fibroblast proliferation. MCL fibroblasts generally did not proliferate significantly more than ACL fibroblasts with the exception of MCL fibroblasts exposed to the highest doses of basic fibroblast growth factor, acidic fibroblast growth factor and platelet-derived growth factor-BB. The data were also compared with those obtained earlier using fibroblasts from skeletally immature rabbits (Schmidt et al., JOR 1995). The proliferative response of both the MCL and the ACL fibroblasts was found to decrease with skeletal maturation. Thus, our findings suggest that animal age and fibroblast origin are important factors in determining the proliferative response to growth factors.

Suppression of antigen-induced arthritis in rabbits by ex vivo gene therapy.

Gene therapy offers a novel approach to treating human joint diseases such as rheumatoid arthritis. In the present study, we have used the retrovirus, MFG-IRAP, to transfer the human IL-1 receptor antagonist protein (IRAP) gene to rabbits' knees and have assessed its impact on inflammatory and chondrodestructive aspects of the acute phase of antigen-induced arthritis in these joints. Surprisingly, intra-articular expression of IRAP was three- to fivefold higher in arthritic knees than in nonarthritic knees, accumulating to levels of over 20 ng/knee in the highest expressing joints. This level of expression produced a marked chondroprotective effect but a milder anti-inflammatory one. Both the increased cartilage matrix catabolism and the inhibition of matrix synthesis that occur in antigen-induced arthritis were abrogated in the presence of the IRAP gene; the latter effect was particularly strong. Of the indices of inflammation that were examined, only leukocyte influx into the joint space was inhibited, and this effect declined with time. Concentrations of rabbit IL-1 were reduced by the IRAP gene, suggesting inhibition of an autocrine induction loop. These data demonstrate that the course of arthritic disease in the rabbit knee can be altered by genetic manipulation, thus encouraging the further development of gene treatments for human joint diseases.

Direct gene delivery to synovium. An evaluation of potential vectors in vitro and in vivo.

OBJECTIVE: To assess the abilities of various vectors to transfer genes to the synovial lining of joints. METHODS: Vectors derived from retrovirus, adenovirus, and herpes simplex virus as well as cationic liposomes and naked plasmid DNA were evaluated. Each construct contained the lac Z marker gene; and one retroviral construct, and one plasmid also contained a gene encoding human interleukin-1 receptor antagonist. Gene expression was under the control of the human cytomegalovirus promoter in all vectors except the retrovirus, where the endogenous 5' long terminal repeat was retained as the promoter. Cultures of rabbit synovial fibroblasts were exposed to these vectors and stained with X-gal to identify lac Z+ cells. Vectors were then injected directly into rabbits' knee joints, and gene transfer and expression were assessed by X-gal staining and polymerase chain reaction (PCR). RESULTS: Adenovirus was a highly effective vector both in vitro and in vivo, with lac Z gene expression persisting for at least 28 days. However, an inflammatory response was noted in vivo. Cells infected in vitro and in vivo with herpes simplex virus also expressed the lac Z gene at high levels, but expression was limited by cytotoxicity. Retroviruses, in contrast, were effective only under in vitro conditions, permitting cell division. Liposomes gave variable in vitro results; when injected into joints in vivo, gene expression was low and was detectable for only a few days, even though a PCR signal persisted for at least 28 days. Unexpectedly, plasmid DNA was captured by the synoviocytes and expressed transiently following intraarticular injection. CONCLUSION: None of the vectors was ideal for in vivo gene delivery to synovium, although adenovirus was clearly the most effective of those tested. Retroviruses, although poor vectors for in vivo gene delivery, are well suited for ex vivo gene transfer to the synovial lining of joints.