Actinidia polygama Water Extract (APWE) Protects Against UVB-Induced Photoaging via MAPK/AP-1 and TGFß-Smad Pathway.

BACKGROUND: Actinidia polygama (silver vine) has been used in oriental medicine to treat gout, rheumatoid arthritis, and inflammation. Actinidia polygama water extract (APWE) is named PB203. OBJECTIVE: To investigate whether PB203 has anti-photoaging effects and to understand the molecular mechanism underlying such effects. METHODS: The antioxidant effect was assessed by 1,1-diphenyl-2-picrylhydrazyl assay and 2',7'-dichlorodihydrofluorescein diacetate staining in ultraviolet B (UVB)-irradiated HaCaT cells with or without PB203 treatment. Type I collagen, matrix metalloproteinase-1 (MMP-1), tissue inhibitor of metalloproteinase (TIMP-1), hyaluronic acid (HA), hyaluronan synthase 1 (HAS1) and HAS2 levels were measuring by enzyme-linked immunosorbent assay or reverse transcription quantitative polymerase chain reaction. Also, we investigate the effects of PB203 on wrinkle formation, and the potential mechanisms underlying such effects were investigated in UVB-induced wrinkle mouse model mice. RESULTS: PB203 alleviated the UVB-induced reactive oxygen species production, phosphorylation of JNK, ERK, and p38, and formation of AP-1. In addition, PB203 inhibited the decreases in type I collagen and TIMP-1 levels, and the increase in MMP-1 levels in UVB-exposed HaCaT cells. In UVB-induced wrinkle mouse model, PB203 inhibited the decreases in elastin and type I collagen levels as well as the increases in MMP-1 expression, wrinkle formation, and skin dehydration. Furthermore, PB203 increased the expression of filaggrin, HAS1, and HAS2, improving the skin barrier function. CONCLUSION: Taken together, we found that PB203 is as a potent candidate to serve as a functional ingredient or therapeutic agent to improve UVB-mediated skin aging.

Anti-Obesity Effects of Artemisia annua Extract in Zucker Fatty Rats and High-Fat Diet Sprague Dawley Rats through Upregulation of Uncoupling Protein 1.

BACKGROUND: Obesity is a widespread disease and is caused mainly by excessive adipocyte differentiation and fat accumulation. Peroxisome proliferation-activated receptor γ (PPARγ) and CCAAT/enhancer-binding proteins (C/EBP) are major components for regulating adipocyte differentiation. Uncoupling protein 1 (UCP1) is a transmembrane protein that can convert white fat to brown adipose tissue. Artemisia annua L. has long been used in East Asia as an herbal drug for anti-oxidant, anti-bacterial, and anti-obesity purposes. METHODS: We investigated the effects of water extracts of A. annua (WEAA) in C3H10T1/2, a mesenchymal stem cell line, by measuring the level of intracellular fat accumulation and the expression of genes associated with adipocyte differentiation. We also evaluated anti-obesity effects of WEAA in Zucker rats, a genetic model for the study of obesity, and in Sprague Dawley rats with high-fat diet (HFD)-induced obesity. RESULTS: In this study, WEAA reduced the expression levels of PPARγ and C/EBPα in C3H10T1/2 cells, as well as the expression of enzymes that regulate fatty acid metabolism. In the Zucker fatty rat model and the HFD-induced obesity rat model, WEAA significantly decreased adipogenic differentiation and white fat accumulation between the scapulae, in contrast to the brown fat that remained unchanged between the groups. A. annua suppressed the expression of the adipocyte differentiation-promoting genes, while increasing the expression of UCP1. CONCLUSION: These results indicated that WEAA could reduce adipocyte differentiation and fat accumulation in in vitro and in vivo model systems, resulting in suppression of obesity and the occurrence of fatty liver due to a HFD.

Efficacy of Artemisia annua L. extract for recovery of acute liver failure.

Artemisia annua L. is an annual herb belonging to the Asteraceae family. It is commonly grown in parts of Asia, including Korea and China, and is called by its nickname Gae-ddong-ssuk, or Chung-ho. The herb is well known for its positive effects on fever and hemostasis, as well as its antibiotic effects. To evaluate the protective properties of A. annua L. on the liver, an acute liver failure animal model was set up with intraperitoneal injection of lipopolysaccharide (LPS) and D-galactosamine (D-galN) in C57BL/6J mice, showing increased levels of AST (aspartate transaminase) and ALT (alanine transaminase). Oral administration of the extract of A. annua L. (EAA) for 2 weeks reduced the level of AST and ALT up to 50% of the levels in the negative control group treated with water vehicle. The efficacy of EAA was more effective than that in a comparative positive control group treated with milk thistle extract. Moreover, EAA protected hepatic cells and tissues from oxidative stresses and inflammatory damages, showing downregulation of inflammatory cytokines such as interleukin-1 beta (IL-1ß), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α). We also found that LPS stimulated the mouse macrophage cell line, Raw264.7, and secreted a tremendous level of proinflammatory cytokines and the secretion of these cytokines was reduced with EAA treatment via downregulation of mitogen-activated protein kinase phosphorylation and p65 translocation. This study demonstrated that A. annua L. extract is a promising treatment for protection against and recovery from liver damage, as well as maintenance of liver health.

The pharmacokinetic and pharmacodynamic properties of site-specific pegylated genetically modified recombinant human interleukin-11 in normal and thrombocytopenic monkeys.

In order to improve the pharmacokinetic and pharmacodynamic properties of recombinant human interleukin-11 mutein (mIL-11) and to reduce the frequency of administration, we examined the feasibility of chemical modification of mIL-11 by methoxy polyethylene glycol succinimidyl carbonate (mPEG-SC). PEG-mIL-11 was prepared by a pH controlled amine specific method. Bioactivity of the protein was determined in a IL-11-dependent in vitro bioassay, its pharmacodynamic and pharmacokinetic properties were investigated by using normal and thrombocytopenic monkey models. N-terminus sequencing and peptide mapping analysis revealed that Lys33 is the PEGylated position for PEG-mIL-11. Bioactivity of PEG-mIL-11 assessed by B9-11 cell proliferation assay was comparable to that of mIL-11. More than 79-fold increase in area-under-the curve (AUC) and 26-fold increase in maximum plasma concentration (Cmax) was observed in pharmacokinetic analysis. Single dose administration of the PEG-mIL-11 induced blood platelets number increase and the effect duration were comparable to that of 7 to 10 consecutive daily administration of mIL-11 to the normal and thrombocytopenic monkey models. PEG-mIL-11 is a promising therapeutic for thrombocytopenia.

[Thrombocytopoietic Efficacy of Pegylated Recombinant Human Interleukin-11 Mutein in Myelosuppressed Mice].

OBJECTIVE: To evaluate the effect of PEGylated IL-11 mutein (PEG-mIL 11) with different dose or injection frequency on thrombocytopenia in myelosuppressed mice and to compare its effect with mIL-11, so as to provide reference data for clinical use. METHODS: Myelosuppressive model with thrombocyopenia was produced in BALB/c mice by whole body 60Co γ-ray irradiation in dose of administration 2.5 Gy followed by i.p. injections of carboplatin at 50 mg/kg. In study of injection frequency, 30 thrombocytopenic BALB/c mice were randomly divided into 5 groups: vehicle control group (once daily on d1, 4, 7), mIL-11 group [200 µg/(kg·d)×9 d], PEG-mIL 11 A group [111800 µg/(kg·d)×1 d (d 1)], PEG-mIL 11 B group 900 µg/(kg·d)×2 d (d 1,5), and PEG-mIL 11 C group [600 µg/(kg·d)×3 d (d 1,4,7)]. The route of administration is subcutaneous injection. The platelet counts were monitored in the subsequant 5 weeks. In study of dose administration, 100 thrombocytopenic BALB/c mice were randomly divided into 5 groups: vehicle control group (once daily on d1 and 5), mIL-11 group 200 µg/(kg·d)×9 d, and PEG-mIL 11 low-, mid-, and high-dose groups (200, 420 and 900 µg/(kg·d)×2 d, once a day on d1 and 5). The route of administration is subcutaneous injection. The platelet counts were monitored every 2-3 days in the subsequant 5 weeks, and CFU-Meg was determined on d 8 of the bone marrow cells collection. RESULTS: After 60Co irradition and carboplatin injection, Plt level decreased with time, and a >80% reduction was noted at nadir when comparing with baseline. In frequency of administration study, the platelet nadir of the 3 PEG-mIL 11 groups were significantly higher than that of vehicle control and mIL-11 groups (P<0.05), while no significant difference was noted among the 3 groups of different administration frequences; In dose level study, the reduction of Plt count at the nadir in the 3 PEG-mIL 11 groups was significantly less than that of vehicle control and mIL-11 groups (P<0.05). And a rapid recovery of Plt count was found in the PEG-mIL 11 groups with a dose-dependent increase of Plt count on d 10. A lower reduction and a rapider recovery of RBC was also found in the PEG-mIL 11 groups. No significant effect on WBC was found for all the treatment groups. An increase in CFU-Meg was observed in PEG-mIL 11 and mIL-11 groups, with higher CFU-Meg in PEG-mIL 11 groups. CONCLUSION: A preventive effect of PEG-mIL 11 on thrombocytopenia in myelosuppressed mice has been confirmed. In comparison with mIL-11, a better effect of PEG-mIL 11 is obtained under lower dose frequency, indicating a better compliance of the treatment regimen, and providing a foundation for developing a long-acting preparation of rhIL-11.

Effect of HX108-CS supplementation on exercise capacity and lactate accumulation after high-intensity exercise.

BACKGROUND: In the present study, we determined the effects of HX108-CS (mixed extract of Schisandra chinensis and Chaenomeles sinensis) supplementation on lactate accumulation and endurance capacity. Furthermore, we examined CK (creatine kinase), LDH (lactate dehydrogenase) activity to determine whether the HX108-CS affected markers of skeletal muscle injury in vivo and in vitro. METHODS: Exercise capacity was measured by an exhaustive swimming test using ICR mice divided into four groups; one group received distilled water (DW) (Control group, n = 10), and the other groups received three different dosages of HX108-CS (10, 50 and 100 mg/kg, n = 10 per group) solution in water orally. Then, for the time-dependent measurements of blood lactate, CK, and LDH, Sprague-Dawley rats were divided into two groups; one received DW (Control group, n = 10), and the other group received HX108-CS (100 mg/kg, n = 10) solution in the same way as mice. Before the exercise test, the animals were given either DW or HX108-CS for 2 weeks. High-intensity treadmill exercise was performed for 30 minutes. Blood samples were collected and analyzed during and after exercise. For the in vitro experiment, C2C12 cells were treated with HX108-CS to examine its effect on lactate production, CK, and LDH activity. RESULTS: Blood lactate concentration was significantly lowered immediately after treadmill exercise in HX108-CS group; however, there were no significant differences in activities of CK and LDH between HX108-CS and control during treadmill exercise and recovery phase. Furthermore, treatment with 100 mg/kg of HX108-CS led to a significant increase in the time to exhaustion in swimming test, and concurrently blood lactate concentration was significantly decreased in 50 and 100 mg/kg treated group. Moreover, our results of in vitro experiment showed that HX108-CS suppressed lactate production, CK, and LDH activity in a dose-dependent manner. CONCLUSIONS: These results suggest that supplementation with HX108-CS may enhance exercise capacity by lowering lactate accumulation. This may in part be related to an amelioration of skeletal muscle injury.

Multicenter, randomized study of genetically modified recombinant human interleukin-11 to prevent chemotherapy-induced thrombocytopenia in cancer patients receiving chemotherapy.

PURPOSE: The aim of this study is to evaluate the efficacy and safety of genetically modified recombinant human IL-11 (mIL-11), using original IL-11 as an active control, in a multicenter randomized trial involving 88 cancer patients undergoing chemotherapy METHODS: Eighty-eight subjects who had platelets ≤ 75 × 10(9)/L during the prior chemotherapy were randomized to the MR or RM group. Cohort MR consists of subcutaneous injection of mIL-11 (7.5 µg/kg/day) for 10 days, beginning 72 h after chemotherapy for a 21-day chemotherapy cycle (cycle-1) followed by that of recombinant human interleukin-11 (rhIL-11) (25 µg/kg/day) for another 10 days (cycle-2). Cohort RM represents the reverse sequence. Intent-to-treat populations of mIL-11 (n = 73) or rhIL-11 (n = 80) were analyzed to evaluate the safety. RESULTS: The incidence of drug-related adverse events of mIL-11 (32.9%) was lower than that of rhIL-11 (51.3%) (p = 0.033). There were no unexpected ≥ grade-3 adverse events, and no subject developed antibodies to the mIL-11 protein. Sixty-two subjects were analyzed for efficacy by measuring average platelet levels. Both mIL-11 and rhIL-11 increased nadir platelet levels (62.6 ± 34.9 × 10(9)/L for mIL-11 vs. 60.2 ± 31.7 × 10(9)/L for rhIL-11) as compared with the untreated control group (41.2 ± 17.7 × 10(9)/L) (p < 0.0001). There was no statistical difference in average platelet levels and platelet recovery rate between mIL-11 and rhIL-11. CONCLUSIONS: This study shows that mIL-11 is well tolerated and has thrombopoietic activity equivalent to one third of the clinical dose of rhIL-11, indicating the potential of mIL-11 for use in the treatment of CIT.

A phase I clinical study of naked DNA expressing two isoforms of hepatocyte growth factor to treat patients with critical limb ischemia.

BACKGROUND: The purpose of the present phase I clinical trial was to evaluate the safety, tolerability, and preliminary efficacy of naked DNA therapy expressing two isoforms of hepatocyte growth factor (pCK-HGF-X7) in critical limb ischemia (CLI) patients. MATERIALS AND METHODS: Twenty-one patients with CLI were consecutively assigned to receive increasing doses (cohort I: 4 mg; cohort II: 8 mg; cohort III: 12 mg; and cohort IV: 16 mg) of pCK-HGF-X7, which was administered into the ischemic calf and/or thigh muscle at days 1 and 15. A safety and tolerability evaluation and measurement of pain severity score using a visual analog scale (VAS), ulcer status, transcutaneous oxygen (TcPO(2) ) and ankle-brachial index (ABI) were performed throughout a 3-month follow-up period. RESULTS: No serious adverse events were observed in any of the 21 patients for the 3-month follow-up period. A significant reduction in pain was observed in the treated patients, with the mean VAS decreasing from 5.95-1.64 (p < 0.001). The mean ABI value increased from 0.49-0.63 (p = 0.026) at 3-month follow-up. The mean TcPO(2) value on the dorsum of the foot, the anterior calf and posterior calf significantly increased from 28.25-39.28 mmHg (p = 0.012), from 22.00-30.63 mmHg (p = 0.046) and 32.05-47.19 mmHg (p = 0.001) at 3-month follow-up, respectively. Wound healing improvement was observed in the six of nine patients that had an ulcer at baseline. CONCLUSIONS: These results support the performance of a phase II randomized controlled trial with pCK-HGF-X7.

Improvement of biological and pharmacokinetic features of human interleukin-11 by site-directed mutagenesis.

Recombinant human interleukin-11 (rhIL-11) has been shown to increase platelet counts in animals and humans and is the only drug approved for its use in chemotherapy-induced thrombocytopenia (CIT). However, due to its serious side effects, its clinical use has been limited. The current work presents significantly improved efficacy of rhIL-11 via knowledge based re-designing process. The interleukin-11 mutein (mIL-11) was found to endure chemical and proteolytic stresses, while retaining the biological activity of rhIL-11. The improved efficacy of mIL-11 was evident after subcutaneous administration of mIL-11 and rhIL-11 in the rodent and primate models. More than three-fold increase in maximum plasma concentration (Cmax) and area-under-the curve (AUC) was observed. Furthermore, three-fold higher increase in the platelet counts was obtained after seven consecutive daily subcutaneous mIL-11 injections than that with rhIL-11. The mIL-11 demonstrated not only improved stability but also enhanced efficacy over the currently used rhIL-11 regimen, thereby suggesting less toxicity.

TNFR-Fc fusion protein expressed by in vivo electroporation improves survival rates and myocardial injury in coxsackievirus induced murine myocarditis.

Tumor necrosis factor-alpha (TNF-alpha) is one of the major cytokines that modulate the immune response in viral myocarditis, but its role has not yet been thoroughly evaluated. We antagonized TNF-alpha using the expressed soluble p75 TNF receptor linked to the Fc portion of the human IgG1 gene (sTNFR:Fc) by in vivo electroporation, and evaluated its effects on experimental coxsackieviral B3 (CVB3) myocarditis. A plasmid DNA encoding sTNFR:Fc (15microg/mouse) was injected into the gastrocnemius muscles of Balb/C male mice followed by electroporation (day -1). Control mice were injected with an empty vector. One day after electroporation, mice were infected with CVB3 (day 0). Serum levels of sTNFR:Fc increased from day 2 and peaked at day 5 following electroporation. The heart virus titers of sTNFR:Fc mice were higher than those of controls at day 3. However, subsequent to day 12, the survival rates of the sTNFR:Fc mice were significantly higher than those of the controls (36% versus 0% at day 27, P<0.01). Histopathological examination indicated that inflammation and myocardial fibrosis were significantly decreased in sTNFR:Fc mice at day 12. The expressed sTNFR:Fc could modulate the inflammatory process during the post-viremic phase of viral myocarditis.

Intrasplenic electro-transfer of IL-4 encoding plasmid DNA efficiently inhibits rat experimental allergic encephalomyelitis.

Most of the previous studies in which cytokine DNA plasmids were delivered by systemic administration exhibited only marginal therapeutic effects, if any, in the EAE model. One strategy to overcome this limitation would be to determine the optimal delivery route leading to significant beneficial effects both in early (prophylactic) and late (therapeutic) treatments. To address this issue, we directly compared the effects of intrasplenic (i.s.) and intramuscular (i.m.) electro-transfer of interleukin-4 (IL-4) DNA in the rat experimental allergic encephalomyelitis (EAE) model. In the preventive experiment, rats received i.m. (25 or 150 microg) or i.s. (25 microg) administration of IL-4 DNA followed by in vivo electroporation the day before MBP immunization. In the late treatment experiment, rats were treated with i.m. (150 microg) or i.s. (25 microg) administration of IL-4 DNA with electroporation 10 days after MBP immunization. As a control the same amount of vector DNA was used. Macroscopic analysis indicated that the onset of moderate to severe EAE in rats treated with i.s. transfer of 25 microg of IL-4 DNA was prevented on a significant level compared with i.m. 25 microg of the IL-4 DNA transfer group or the control group in the preventive experiments. More importantly, i.s. transfer of 25 microg of IL-4 DNA considerably suppressed the severity of EAE in late treatment experiments while i.m. transfer of 150 microg of IL-4 DNA had little effect. The MBP-specific expression of IFN-gamma from stimulated splenocytes was considerably decreased by the i.s. IL-4 DNA transfer group both in the preventive and therapeutic experiments while i.m. transfer had this effect only in the preventive protocol. Histological analysis showed that spinal cord inflammation was considerably reduced in the i.s. IL-4 DNA transfer group. These data provide the first demonstration that i.s. electro-transfer of IL-4 DNA is more effective both in the prevention and modulation of EAE than i.m. transfer and that i.s. electro-gene transfer may present a new approach to cytokine therapy in autoimmune diseases.

Electrotransfer of human IL-1Ra into skeletal muscles reduces the incidence of murine collagen-induced arthritis.

BACKGROUND: It has previously been demonstrated that high levels of gene expression in skeletal muscles can be achieved after direct in vivo electrotransfer of naked plasmid DNA. The purpose of this study is to examine the potential of in vivo electroporation of plasmid DNA encoding human IL-1Ra for the prevention of murine collagen-induced arthritis (CIA). METHODS: DBA/1 mice were injected in gastrocnemius muscles with plasmid DNA followed by in vivo electroporation. To uncover the optimum conditions of gene transfer, various electric field strengths and different amounts of plasmid DNA were applied. Calf muscles around the injected areas were investigated with histological methods for damage to muscle tissue. The levels of human IL-1Ra expression in the injected area and also in the serum were determined with ELISA for human IL-1Ra. Based on these data, the effects of electrotransfer of plasmid DNA were tested using the murine CIA model. DBA/1 mice were immunized with bovine collagen type II at the base of the tail. On day 21, mice were given a booster injection with the same antigen. Mice were divided into two groups on day 26. One group of mice received plasmid containing the IL-1Ra cDNA sequence, while control mice were given plasmid lacking the IL-1Ra coding sequence. The incidence of arthritis was evaluated by macroscopic analysis, histological analysis, and the levels of inflammatory cytokines. RESULTS: IL-1Ra expression increased as a function of the electrical field strength and the amount of DNA. 200 V/cm (eight pulses; 20 ms per pulse; 1 Hz) and 15 microg of plasmid DNA per mouse were found to be optimum for gene transfer. After in vivo electroporation, gene expression in both muscle and serum increased gradually, reaching a peak value on day 10. Significant levels of human IL-1Ra expression were maintained for 20 days. Macroscopic analysis showed that the onset of CIA was significantly inhibited by direct electrotransfer of plasmid DNA encoding human IL-1Ra. Histological analysis of knee joints showed that the incidence of arthritis in knee joints was also prevented. The levels of mouse IL-1beta and IL-12 in paws were significantly lower in the group treated with IL-1Ra than those in the control group. CONCLUSIONS: These results demonstrate that direct electrotransfer of plasmid containing the human IL-1Ra cDNA sequence to skeletal muscle can reduce the incidence of CIA in mice.

Protection against collagen-induced arthritis by electrotransfer of an expression plasmid for the interleukin-4.

Rheumatoid arthritis (RA) is a chronic inflammatory joint disease, leading to cartilage and bone destruction. We investigated whether the electrotransfer of IL-4 DNA could regulate the disease progress of murine collagen-induced arthritis (CIA). The maximum serum level of mIL-4 was measured by 340 pg/ml on day 1 following DNA transfer. The onset of severe CIA and the degree of synovitis and cartilage erosion were significantly reduced in mice treated with IL-4 DNA (P<0.05). The beneficial effect of IL-4 gene transfer lasted for at least 17 days subsequent to treatment. The expression of IL-1beta was considerably decreased in the paws by IL-4 DNA transfer (P<0.01). On the contrary, the ratio of TIMP2 to MMP2 significantly increased in the IL-4 DNA-treated group (P<0.01). These data demonstrated that electroporation-mediated gene transfer could provide a new approach as an IL-4 therapy for autoimmune arthritis.

Local expression of interleukin-1 receptor antagonist by plasmid DNA improves mortality and decreases myocardial inflammation in experimental coxsackieviral myocarditis.

BACKGROUND: The inflammatory cytokines have an important role in the pathogenesis of viral myocarditis. Inerleukin-1 (IL-1) is one of the major cytokines that modulate the outcome of viral infection. Among the methods for in vivo gene transfer, direct injection of plasmid DNA is one that is simple and feasible. In this study, we expressed human IL-1 receptor antagonist (hIL-1Ra) in the mouse heart by direct injection of a novel plasmid vector and evaluated its effects on coxsackieviral (CVB3) myocarditis. METHODS AND RESULTS: A plasmid vector expressing hIL-1Ra (total 40 microg/mouse) was injected into the heart apex of 8-week-old inbred female Balb/C mice (day 3). On day 0, mice (IL-1Ra-CVB3, n=35) were infected intraperitoneally with 10(4) PFU of CVB3; control mice (pCK-CVB3, n=15) were injected with empty vector on day -3 and infected on day 0. hIL-1Ra was expressed in the heart, reached its peak on day 5, and persisted for 2 weeks. The 14-day survival rate of IL-1Ra-CVB3 was higher (77%) than that of controls (30%, P<0.01). Myocardial virus titers on day 3 were lower in IL-1Ra-CVB3 mice. Myocardial inflammation on day 7 and fibrosis on day 14 were markedly decreased in IL-1Ra-CVB3. CONCLUSION: These results showed that direct injection of the expression plasmid vector into the heart was an effective method to transfer the cytokine gene in vivo, and expressed IL-1Ra in the heart can modulate the deleterious effect of the host immune response in viral myocarditis.

Angiostatin gene transfer as an effective treatment strategy in murine collagen-induced arthritis.

OBJECTIVE: To determine the efficacy of local therapy with human angiostatin gene in murine collagen-induced arthritis (CIA). METHODS: DBA/1 mice were immunized with bovine type II collagen. Before the onset of arthritis, NIH3T3 fibroblasts, transduced with angiostatin-expressing retroviral vectors or control vectors, were transplanted into the knee cavity. The incidence of arthritis in the knee joints was evaluated histologically based on pannus formation and cartilage destruction. Paws were evaluated macroscopically for redness, swelling, and deformities and immunologically for levels of interleukin-1 beta. Angiogenesis in paws and knee joints was studied by immunohistochemistry using anti-CD31 antibody and measurement of von Willebrand factor levels. RESULTS: Pannus formation and cartilage erosion were dramatically reduced in knees transplanted with angiostatin-expressing cells. In addition, the onset of CIA in the ipsilateral paws below the knees injected with the angiostatin gene was significantly prevented. Furthermore, angiostatin gene transfer inhibited arthritis-associated angiogenesis. CONCLUSION: Local production of angiostatin in the knee was able to prevent the onset of CIA not only in the knee injected with genetically engineered cells, but also in the uninjected ipsilateral paw. This suggests that transfer of the angiostatin gene, and potentially also its protein, may provide a new, effective approach to the treatment of rheumatoid arthritis.