Endostatin Gene Transfer Inhibits Joint Angiogenesis and Pannus ...

doi:10.1006/mthe.2002.0590, available online at http://www.idealibrary.com on IDEAL

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Endostatin Gene Transfer Inhibits Joint Angiogenesis and Pannus Formation in Inflammatory Arthritis Guoyong Yin,1 Weimin Liu,2 Ping An,1 Ping Li,3 Ivan Ding,2 Vicente Planelles,4 Edward M. Schwarz,5 and Wang Min1,* 1

Center for Cardiovascular Research, 2Department of Radiation Oncology, 3Department of Microbiology & Immunology, 4Medical Oncology and Microbiology & Immunology, 5Center for Musculoskeletal Research, University of Rochester Medical Center, 601 Elmwood Avenue, Box 679, Rochester, New York 14642, USA *

To whom correspondence and reprint requests should be addressed. Fax: (716) 275-9895. E-mail: [email protected].

Rheumatoid arthritis is a prevalent example of an inflammatory angiogenic disease, which is mediated by pro-inflammatory and pro-angiogenic cytokines such as tumor necrosis factor (TNF). To evaluate the effect of the potent anti-angiogenic factor endostatin on TNF-induced inflammatory arthritis, we injected an endostatin-expressing lentiviral vector directly into the joints of human TNF-transgenic mice before the onset of disease. Histological analysis of the injected joints 8 weeks later revealed that endostatin reduced blood vessel density within the synovial tissues and an overall mean arthritis index. In vitro and in vivo examination of the potential mechanism by which endostatin inhibited the arthritis revealed that endostatin blocks TNF-induced activation of JNK and JNK-dependent pro-angiogenic gene expression. These data suggest a novel mechanism by which endostatin inhibits angiogenesis, and demonstrates the potential utility of anti-angiogenic gene therapy for treatment of inflammatory arthritis. Key Words: rheumatoid arthritis, inflammation, angiogenesis, antiangiogenesis, endostatin, TNF-, JNK, gene therapy, lentiviral vector, chemokine

INTRODUCTION Angiogenesis is a key component of inflammatory diseases such as rheumatoid arthritis (RA) [1,2]. RA is a prevalent example of an inflammatory angiogenic disease affecting 1% of the population [1–4]. RA progression involves the thickening of the synovial lining due to proliferation of fibroblast-like synoviocytes (FLS) and infiltration by inflammatory cells in response to pro-inflammatory cytokines [4]. This proliferative mass, the pannus, invades and destroys articular cartilage and bone, leading to irreversible destruction of joint structure and function. Although multiple cytokine signaling systems with redundant effects on angiogenesis are known to be operant in RA and other inflammatory diseases, several lines of evidence support that tumor necrosis factor (TNF) is a critical cytokine in the pro-inflammatory and pro-angiogenic cascades [4]: 1) TNF is expressed at high levels in inflamed synovium [5]; 2) addition of anti-TNF antibodies inhibits the production of other pro-inflammatory cytokines such as IL-1, IL-6, IL-8, and GM-CSF [6]; 3) TNF transgenic mice develop a chronic erosive arthritis [7]; 4) virtually all animal models of arthritis are ameliorated by anti-TNF [8]; and 5) several human clinical trials of anti-TNF therapy for RA have shown beneficial results [9]. Based on this

MOLECULAR THERAPY Vol. 5, No. 5, May 2002, Part 1 of 2 Parts Copyright © The American Society of Gene Therapy 1525-0016/02 $35.00

paradigm, human TNF- transgenic mice (TNF-Tg), along with collagen-induced arthritic animals and KRN-NOD transegnic mice, are commonly used models of RA [7,10,11]. Beginning at 3 weeks of age and continuing throughout varying developmental stages, hyperplasia of the synovial membrane, neovascularization, and polymorphonuclear and lymphocytic inflammatory infiltrates of the synovial space can be seen in nearly all joints examined in the TNF-Tg mice. Pannus formation, articular cartilage destruction, and massive destruction of fibrous tissue are observed in the advanced stages of disease. These symptoms all closely mimic those of human disease. Endothelial cells (EC) are among the principal physiological targets of pro-inflammatory cytokines like TNF, which uses receptors and associated adapter proteins to trigger multiple signaling pathways leading to activation of JNK and NF-KB as well as other pathways [12]. We and others have studied the biological significance of these signaling pathways in EC [13,14]. Activation of JNK is also required for TNF-induced gene expression of pro-angiogenic molecules including VEGF, bFGF, IL-8, and MCP-1 [15]. Endostatin is a 22-kDa carboxy-terminal fragment of collagen XVIII that is present in circulation of normal and

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tumor-bearing individuals [16]. It is a specific inhibitor of endothelial cell proliferation, migration, and angiogenesis [16,17]. It has been shown that endostatin can inhibit systemic angiogenesis and tumor growth in mouse models [16]. We have expanded these findings in demonstrating that endostatin gene transfer is also an effective method of inhibiting systemic angiogenesis and tumor growth in vivo [18–20]. The similarity of the inflammation and angiogenesis involved in tumor and pannus growth, in cancer and RA, respectively, prompted us to examine the effects of endostatin on joint angiogenesis and pannus formation in TNF-Tg mice. For these studies we chose to use the replication-defective HIV vector [21], as this vector has several advantages for this kind of gene therapy [22]. Here we show that direct injection of an endostatin-expressing lentiviral vector into joints of diseased mice inhibited angiogenesis and synovial hyperplasia, and ameliorated the progression of the erosive arthritis. Furthermore, we show that endostatin inhibited TNF-induced JNK activation and expression of the JNK-dependent angiogenic genes VEGF, bFGF, IL-8, and MCP-1, suggesting a novel mechanism of anti-angiogenesis by endostatin. These studies indicate that endostatin gene therapy may be a useful treatment for patients with RA.

RESULTS Endostatin Expression in Vitro and in Vivo in an HIV-Derived Lentiviral System To develop a secreted endostatin molecule that can be delivered by gene therapy, we added the coding sequence of the secretion signal from the mouse Ig chain with a HA tag to the amino terminus of the mouse endostatin coding sequence [18]. We constructed a dual cDNA expression cassette, lentiviral vector to co-express a secreted form of ES (Ig-HA-ES) and the enhanced green fluorescent protein (EGFP) from a bicistronic mRNA, linked by an internal ribosome entry sequence (IRES). A diagram of the expression cassette (HIV-ES-EGFP) is shown (Fig. 1A). Because EC and FLS are the two major resident cell types within the pannus, we examined the lentiviral vector transduction efficiency in these two cell types in vitro. We infected 1 106 EC (human umbilical vein EC, HUVEC) or FLS from the TNF-Tg mice with HIV-ES-IRES-EGFP at a multiplicity of infection (MOI) of 50, 10, and 1. We visualized GFP-positive cells by fluorescence microscopy; both cell types were transduced at efficiencies of 100% at MOI = 50, 50% at MOI = 10, and 1% at MOI = 1. To evaluate endostatin expression in these transduced cells, we carried out western blot analysis with cell culture medium. Both EC and FLS cultures produced secreted endostatin protein, detected as a single band with the predicted molecular weight of 22 kDa (Fig. 1B). We also determined by ELISA that 1.5 g/ml of endostatin was expressed in the culture medium of the transduced cells (106 cells at MOI = 50).

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FIG. 1. Expression of endostatin in vitro and in vivo. (A) Schematic representation of the endostatin expression vector in an HIV-derived lentiviral system. CMVe/p, CMV enhancer/promoter; Ig, signal sequence from murine Ig chain gene; HA, an epitope tag; IRES, internal ribosomal entry sequence; EGFP, enhanced green fluorescence. (B) In vitro expression in HUVEC (EC) and fibroblast-like synoviocytes (FLS) after lentiviral infection. EC or FLS were infected with empty vector (VC) or endostatin (ES)-expressing lentivirus. Condition media were harvested at 24 hours and endostatin was determined by western blot using anti-HA antibody. (C). In vivo expression in serum. The empty vector (VC) or endostatin (ES) lentivirus was injected into joints of TNF-Tg mice and endostatin protein was determined by immunoprecipitation followed by western blot with anti-HA. Data shown are from day 14. Lane 1, 2 ng of endostatin expressed by HUVEC infected in vitro was added to a control serum before immunoprecipitation and western blot (STD); lanes 2–4, serum from three individual mice injected with endostatin-expressing lentivirus (ES); lanes 5–7, serum from three individual mice injected with the empty vector (VC). Endostatin and its degraded products are indicated.

Next we evaluated endostatin expression from the HIV-ES-IRES-EGFP vector in vivo. We injected a single dose of 2 106 pfu HIV-ES-IRES-EGFP or HIV-IRES-EGFP (empty vector control) into the joint of TNF-Tg mice (three mice per group) and measured endostatin expression in the serum on days 7 and 14 by immunoprecipitation with anti-HA followed by western blot with anti-HA antibodies. To determine the relative amount of endostatin expressed in the serum, we added various amounts of recombinant endostatin protein expressed by HUVEC in vitro (1, 2, 5, and 10 ng) to the control serum before immunoprecipitation and western blot. We found that endostatin was expressed in the serum of all three mice in the HIV-ES-IRES-EGFP group, but not in any of the emptyvector-treated group. Endostatin was expressed on day 14 at a concentration of approximately 40 ng/ml (Fig. 1C), but decreased to a basal level on day 28. The endostatin expressed in serum is approximately the same size as that expressed by HUVEC infected in vitro, although degradation products were clearly visible by western blot. Endostatin Expression Inhibited Joint Angiogenesis and Prevented Joint Destruction We evaluated the effect of endostatin gene therapy on

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FIG. 2. Endostatin inhibits pannus formation and joint angiogenesis. The empty vector (VC) or endostatin-expressing lentivirus (ES) was directly injected into joints of TNF-Tg mice three times with an interval of 2 weeks. At 4 weeks after the third injection, ankles were harvested for histology and immunohistochemistry analyses. A total of five mice per group, two joints per mouse, and two sections per joint (n = 20) were analyzed. Representative joints receiving an injection of empty vector (VC) or endostatin-expressing lentivirus (ES) are shown. (A) Localization of transgene expression. GFP-positive cells were visualized under fluorescence microscope (left). The corresponding hematoxylin and eosin staining is shown on the right. GFP-positive cells are observed within the pannus around an injection site (shown by arrows). GFP-positive cells were not present in non-injected joints. (B) Hematoxylin and eosin stained sections from the treated mice demonstrating the efficacy of ES gene therapy in preventing arthritis. Of note is the difference in the size of the pannus (P) and the extent of erosion of the articular surfaces (A), in the ES versus VC treated mice. (C) Immunohistochemistry of CD31 for blood vessels. Vessel profiles are indicated by arrows. (D) Pannus formation was graded (grade 0–5) as described in the text. (E) Vessel density was quantified and data are presented as vessel density (number of vessel/mm2 pannus). Errors are SD. *Significant difference from empty vector group, P < 0.05.

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inflammatory arthritis by directly injecting HIV-ES-IRESEGFP into the joints of TNF-Tg mice at the time of disease onset (2 months). The empty vector (VC) or endostatinexpressing lentivirus (ES) was injected three times with an interval of 2 weeks. At 4 weeks after the third injection, we harvested the ankles of the mice for histology and immunohistochemistry analyses. No bleeding and obvious


damages to organs (liver, lung, heart, and spleen) were observed in the treated animals when joint tissues were harvested. First, we determined the relative in vivo transfection efficiency by examining GFP-positive cells under fluorescence microscopy. GFP was readily detectable in the cytoplasm of nucleated cells at the injection site, but not in the uninjected joints (Fig. 2A). The kinetics and level of endostatin expression were not significantly altered by repeated injections, suggesting that no immune response to endostatin protein produced from the transgene was elicited. All mice in the untreated (data not shown) or VC-treated groups showed severe disease involving synovial hyperplasia, loss of articular surfaces, and destruction of the subchondral bone (Fig. 2). Using a modified mean arthritis index (MIA) to evaluate the joint destruction in these mice, we found that all joints of VC-treated mice had severe arthritis (grade 4 ± 1; Figs. 2B and 2D). Extensive angiogenesis was also evident in the inflammatory tissue of the arthritic joints, as the number of CD31-positive blood vessels in the synovium increased compared with that of age- and sex-matched non-transgenic littermates (Fig. 2C). In contrast, mice given the HIV-ES-IRES-EGFP treatment displayed significantly reduced pannus formation and joint destruction (grade 1.5 ± 1.2; n = 5, P < 0.05; Figs. 2B and 2D). Endostatin decreased the number of vessel profiles consistent with an inhibition of the joint angiogenesis (Figs. 2C and 2E). Statistical analysis indicated that vessel density (number of vessels/mm2 pannus) was reduced by 3.2fold (VC = 24 ± 6/mm2, ES = 7.5 ± 2/mm2; Fig. 2E). In all experiments, we injected the virus into the right knee and right ankle in the same mouse. However, we observed that

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there were no significant differences among directly injected joints (right knees and ankles) and noninjected joints (left knees and ankles) in the HIV-ES-IRES-EGFPtreated mice. GFP-positive cells were detected only in the injected joints but not in the noninjected joints, suggesting that the injected DNA was only located at the site of injection. This is consistent with our previous observation that endostatin expressed in local tissues can be released into the circulation and can elicit systemic inhibition of angiogenesis [18–20].

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FIG. 3. Endostatin inhibits TNF-induced JNK but not NF-B activation in EC. (A) Endostatin inhibits JNK activation in EC. HUVEC were infected with empty vector or endostatin-expressing lentivirus. EC were either untreated or treated with TNF for 15 minutes. JNK activation was determined by an in vitro kinase assay as described [14]. JNK1 and JNK2 proteins were determined by western blot. (B) Endostatin has no effect on NF-B activation in EC. EC (HUVEC) infection and TNF treatment were performed as in (A). NF-B activation was determined by EMSA using B probe as described [14]. (C) Endostatin inhibits a JNK reporter gene. HUVEC were transiently transfected with the VC or ES plasmids together with a JNK promoter-reporter gene. Relative luciferase activities (luciferase/ranilla unit) from untreated or cytokine-treated samples are presented from mean of duplicate samples. Errors are SEM. Similar results were obtained from two additional experiments.

Endostatin Inhibits TNF-Induced JNK Activation in EC TNF induces gene expression of many molecules involved in inflammation, angiogenesis, and proliferation by inducing activation of JNK [23]. We hypothesized that endostatin inhibits angiogenesis and arthritis by blocking TNF signaling in EC. We analyzed the effects of endostatin on TNF-induced activation of JNK using an in vitro kinase assay and NF-B activation by electrophoretic mobility shift assay (EMSA) as we have done previously [14]. Consistent with our previous findings [14], TNF strongly activated JNK and NF-B in EC (Fig. 3). However, EC infected with HIV-ES-IRES-EGFP displayed decreased TNFinduced JNK activation with no effects on JNK protein expression (Fig. 3A). In contrast to JNK, TNF-induced NF-B activation was unaffected in these cells (Fig. 3B), suggesting specificity of endostatin for the JNK pathway. Activated JNK phosphorylates transcription factors c-Jun and ATF-2 leading to enhanced JNK-dependent gene expression [23]. To see if endostatin had effects on JNK-dependent gene expression, we carried out transient transfection assays with a JNK-dependent reporter gene construct. Activity of the JNK-dependent reporter gene (luciferase) in EC was increased sixfold in response to TNF (Fig. 3C). However, we failed to detect this induction in the endostatin-expressing cells. Consistent with the EMSA results, endostatin had no effect on NF-kB reporter gene activity (data not shown). Our data also showed that endostatin had no effect on JNK or NF-B reporter gene activity in FLS, consistent with previous reports that endostatin responses are specific to EC. Endostatin Inhibits Expression of JNK-Dependent Pro-angiogenic Genes in EC JNK activation is important for TNF-induced expression of pro-angiogenic genes such as VEGF, bFGF, IL-8, MCP-1, and MMPs [15,24]. To determine if endostatin inhibits the endogenous expression of these pro-angiogenic genes in EC, we measured expression of IL-8 and MCP-1 in endostatin-expressing cells. We determined expression of IL-8 and MCP-1 by RNase protection assays (Fig. 4A). The results show that the amounts of IL-8 and MCP-1 mRNA induced by TNF stimulation of EC were decreased by 55% and 65%, respectively, in the endostatin-expressing cells (Fig. 4B).



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significantly alter CD31 mRNA level within 36 hours postinfection (Fig. 5C). Quantitation analyses from three independent experiments using CD31 for normalization indicate that endostatin inhibited expression of VEGF, bFGF, MIP-2, and MCP-1 by 55%, 35%, 45%, 65%, and 32%, respectively (Figs. 5D and 5F). In contrast, no effects of endostatin on FLT-1, FLT-4, TIE-1, and Rantes mRNA were observed, indicating a specificity of endostatin for JNKdependent, pro-angiogenic molecules (Figs. 5C and 5E). Endostatin significantly increased mRNA expressions of thrombospondin-1 (TSP-1, by twofold; Fig. 5D), a potent anti-angiogenic factor [28].

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FIG. 4. Endostatin inhibits TNF-induced expression of pro-angiogenic genes in EC. (A, B) RNase protection assay for MCP-1 and IL-8. HUVEC were infected with VC or endostatin-expressing lentivirus. At 24 hours postinfection, EC were treated with TNF (10 ng/ml) for 2 hours. Total RNA was isolated for RNase protection assay. (A) Representative of two experiments is shown. (B) MCP-1 and IL-8 mRNAs were normalized with L32. Relative levels of gene expression (compared with VC) are presented from mean of two experiments. Errors are SEM.

Endostatin Inhibits JNK Activity and JNK-Dependent Expression of Pro-angiogenic Genes in RA Mice To determine if endostatin inhibits TNF-induced JNK activation and JNK-dependent expression of, for example, chemokines and angiogenic factors, which have been implicated in RA progression [15,25–27], we examined the effects of endostatin expression on JNK activity, and expression of MIP-2 (mouse homolog of human IL-8), and MCP-1 in arthritic mice. TNF-Tg mice received 2 106 pfu of empty vector or HIV-ES-IRES-EGFP and the injected joints were harvested at the indicated time for JNK kinase assay, RNase protection assays for chemokines. JNK activity was higher in the diseased mice compared with the normal control (Fig. 5A). Endostatin significantly decreased JNK activity in the disease mice at 24 hours postinjection with no effect on JNK protein expression (Fig. 5B). RNase protection assays showed that endostatin decreased mRNA expression of VEGF, bFGF, MIP-2, and MCP-1 at 36 hours postinjection (Figs. 5C–5F). CD31 mRNA is abundant in joints and endostatin did not


Research over the past decade has demonstrated the involvement of angiogenesis in the pathogenesis of RA, which is the most prevalent example of inflammatory angiogenic disease in people [1,2]. Here we have shown that the specific anti-angiogenic factor endostatin inhibits joint angiogenesis leading to suppression of pannus formation and arthritic progression in the TNF-Tg mouse model, suggesting a critical role of angiogenesis in RA progression. We also provide evidence that the mechanism by which endostatin acts is through the inhibition of JNK activation, resulting in the downregulation of the proangiogenic factors VEGF, bFGF, IL-8, and MCP-1. It has been suggested that suppression of joint angiogenesis could be used to treat patients with RA. The antiangiogenic small molecule fumagillin derivative AGM (TNP-470) prevented arthritis and reversed established disease in both rat and mouse models [29,30]. Taxol, which can induce EC apoptosis, was also effective in an animal model of RA [31]. It has been shown that intra-articular administration of a cyclic peptide antagonist of integrin av3 resulted in inhibition of synovial angiogenesis and reduced synovial cell infiltrate, pannus formation, and cartilage erosions in a rabbit antigen-induced RA model [32]. These studies demonstrate that anti-angiogenesis therapy provides an efficient approach to treat RA. However, prolonged treatment is required for anti-angiogenic therapy in chronic diseases such as RA and cancer [33]. Gene therapy would be an ideal approach for this purpose. As we and others previously demonstrated, anti-angiogenic gene therapy has advantages over the recombinant protein approach [18–20,34–36]. Gene therapy has been recently applied to RA by intra-articular injection of adenoviral expression vector for p53 [37]. Here, we expressed endostatin in an HIV-derived lentiviral system that has low immunogenicity and high transduction efficiency [38]. A single injection of HIV-ES-IRES-EGFP produces ES in a biologically active form that is detectable in the circulation for up to 2 weeks. Endostatin expression ameliorated the progression of inflammatory arthritis in TNF-Tg mice. Our study substantiates further the development of anti-angiogenic gene therapy for inflammatory arthritis.

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Endostatin is a member of an expanding family of antiangiogenic factors. Other members include angiostatin, thrombospondin-1 (TSP-1), maspin, and interferon. However, the mechanism by which they exert their angiostatic function is largely unknown. Recent studies indicate that TSP-1 induces anti-angiogenesis by activating caspase 3 via a CD36 receptor and Src kinase-dependent pathway [28]. IFN- inhibits production of angiogenic factor bFGF in skin tumor cells [39]. We have shown that endostatin exerts its anti-angiogenic function by a novel mechanism, that is, by modulating TNF

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FIG. 5. Endostatin inhibits JNK activity and JNK-dependent gene expression in RA diseased mice. TNF-Tg mice were treated with empty vector (VC) or endostatin-expressing lentivirus (ES). Viral injection, tissue harvest, JNK kinase assay, and RNase protection assays were performed as described. (A) JNK activity is increased in TNF-Tg mice compared with wild-type C57bl. Two joints from each mouse were harvested in lysis buffer and total 40 g protein was used for JNK expression and kinase assay using 10 g GST-c-Jun as a substrate. (B) Endostatin inhibits JNK activity. Two joints from each mouse were harvested at 24 hours postinjection and combined for JNK assay as described above. JNK protein was determined by western blot with antiJNK1 (Santa Cruz). (C, E) RNase protection assay for pro-angiogenic and antiangiogenic factors. Joints were harvested at 36 hours postinjection. We used 30 g total RNA from four joints in each group (two mice per group and two joints from each mouse) for RNase protection assay. The tRNA was used as a control. The probes are indicated on the left and the protected fragment for each gene is labeled on the right. (D, F) Quantitation of the mRNA of angiogenic factors and chemokines. mRNA expression levels were measured with a Phosphorimager and were normalized with the CD31 mRNA level. Data are presented as a ratio (number above each bar) of an angiogenic factor mRNA level from ES-treated mice compared with VC group and are means (± SD) from three independent experiments (total six mice per group). *Significant difference from empty vector group, P < 0.05.

signaling in arthritic joints. It has been established in RA patients that TNF triggers inflammatory and angiogenic cascades. The angiogenic activity of TNF is likely mediated by its multiple actions on EC. JNK activation and JNK-dependent gene expression have been implicated in progression of RA [25,26]. In our study, TNF-induced JNK activation and JNK-dependent gene expression of angiogenic molecules were specifically inhibited by endostatin, highlighting the importance of JNK activation in RA progression. VEGF is a potent angiogenic factor in RA, and an imbalance in production between VEGF and endostatin has been found in RA



patients [40]. It has also been shown that inhibition of VEGF using either soluble VEGF receptor or anti-VEGF antibody ameliorated murine collagen-induced arthritis [41–43]. Our data show that endostatin inhibited VEGF expression in arthritic joints, suggesting that endostatin inhibits joint angiogenesis and arthritis progression in part by suppressing VEGF production. Reduction of chemokine and cytokine production by activated EC has been suggested to be one of the mechanisms by which anti-angiogenesis treatment blocks RA progression [44]. We show that endostatin decreases expression of the JNK-dependent pro-angiogenic molecules VEGF, bFGF, IL-8 (MIP-2 in mouse), and MCP-1 in EC in vitro and in vivo. EC is abundant in the diseased joints visualized by both CD31 immunohistochemistry and CD31 mRNA expression. Furthermore, mounting evidence shows that the action of endostatin is EC-specific [16,17]. Thus, although we cannot exclude the possible effects of endostatin on infiltrated leukocytes, which are known to contribute significant JNK activity in arthritic joints [25,26], our data highlight a dominant role of EC-derived, JNK-dependent, pro-angiogenic molecules in arthritis. Chemokines such as MCP-1 and IL-8 have proinflammatory and pro-angiogenic activities, suggesting that endostatin may exhibit anti-inflammatory function. It is well known that a poor correlation between synovial inflammation and joint destruction has been observed in RA and effective anti-inflammatory reagents often have little or no influence on disease progression [2,44]. Thus, the inhibitory effect of endostatin on RA progression is in large part due to its anti-angiogenesis activity. We have previously shown that endostatin increases the anti-angiogenic peptide TSP-1 in tumor models [20]. In consistence with this observation, we have shown that endostatin also increases expression of TSP-1 in RA mice. These data suggest that endostatin may regulate the balance of angiogenic and anti-angiogenic factors. Our study demonstrates that joint neovascularization has an important role in the progression of arthritic disease, and suggests that anti-angiogenic gene therapy is a valid approach for therapeutic intervention for patients with inflammatory arthritis.

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Cells and cytokines. Human umbilical vein endothelial cells (HUVEC) were isolated and cultured as described [14]. Human rTNF was from R&D Systems, Inc. (Minneapolis, MN) and used at 10 ng/ml. Lentiviral vector construction, production and assay. The coding sequence of the secretion signal from the mouse Ig chain with a HA tag to the N terminus of the mouse endostatin coding sequence [18] was inserted into a dual cDNA expression cassette vector to co-express endostatin with EGFP, on a bicistronic mRNA, linked by IRES in the pHR-CMV (a gift from Inder Verma, The Salk Institute for Biological Studies, La Jolla, CA). The viral vector was prepared as described [38]. Briefly, 1.5 106 293 cells were plated in 10-cm plates, and transfected the following day with 12.5 g of pCMVR8.2 VPR, 12.5 g of the pHR-GFP plasmid, and 5 g of pVSVG, by calcium phosphate DNA precipitation. Conditioned medium


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was harvested 62 hours after transfection, cleared of debris by low-speed centrifugation, filtered through 0.45-M filters (Falcon, Lincoln Park, NJ), and assayed for transduction of HUVEC, by infecting cells overnight with serial dilutions of vector stock in culture medium supplemented with 8 g/ml Polybrene (Sigma, St. Louis, MO). After medium replacement, the cells were further incubated for 36 hours, and expression of GFP was scored by microscopy. The ES expression was determined by western blot. Concentrated vector stocks were prepared by ultracentrifugation of conditioned medium at 50,000g for 90 minutes. The pellet was resuspended in 0.05% of the starting volume in sterile PBS containing 4 g/ml Polybrene. Stocks were titered as described above and stored frozen at –80 C. ELISA. The concentration of endostatin from cell culture was quantified by Accucyte Endostatin (Cytimmune Science Inc., College Park, MD) as described [18]. Animal models and treatment. The 3647 line of the TNF-Tg mice (C57B/c background) was used with non-transgenic littermates as controls. We injected 2 106 pfu of HIV-ES-IRES-EGFP into 2-month-old mice in a volume of 30 l into right joints (10 l to an ankle and 20 l to a knee). A single injection was used for expression of endostatin, JNK kinase and RNase protection assays. Three injections with an interval of 2 weeks were applied for efficacy studies. Mice joints were harvested at indicated times. Guidelines for the humane treatment of animals were followed as approved by the University Committee on Animal Resources. Histology. Tissues from mice were used in morphology and immunolabeling studies as described [45]. The tissues were fixed in Bouin’s fixative overnight, decalcified for 2–4 days and embedded in paraffin. The tissue was then sectioned and stained with hematoxylin and eosin. The mean arthritis index (MAI) was scored as follows: 0, normal; 1, synovial hyperplasia; 2, mild destruction of articular surface; 3, severe destruction of articular surfaces; 4, mild subchondral bone loss; and 5, severe subchondral bone loss. The data for the effect of ES gene therapy on MAI were analyzed by ANOVA. In all cases a P value of < 0.05 was considered to be statistically significant. Immunohistochemistry and vessel density. Immunohistochemistry for CD31 was carried out as described [18]. Joint vessels are identified by immunohistochemistry with an antibody against the endothelial marker CD31 (Santa Cruz Biotechnology Inc., Santa Cruz, CA). Thin-walled, capillary-like vessels were counted. Vessel density was defined as number of vessels/mm2 pannus. Data for the effect of endostatin gene therapy on joint vessels were analyzed by ANOVA. In all cases a P value of < 0.05 was considered to be statistically significant. RNase protection. At 24 hours postinfection, HUVEC were treated with TNF for 2 hours. For mice, virus-infected joints were harvested at 36 hours postinfection. Total RNA was isolated for RNase protection assay according to the manufacturer’s protocol (PharMingen, San Diego, CA). Cells or tissues from each treatment group were isolated by pulverizing the frozen and dissolving it in TRIzol reagent (MRC, Cincinnati, Ohio). RNase protection was performed using established multiprobe template sets (PharMingen) as described [20]. For angiogenic and anti-angiogenic factors, a RiboQuant Custom Mouse Template Set was used. For chemokines, human CK5 was used for HUVEC and mouse CK5 for mice samples. The quantitation of mRNA expression level tested for each sample was measured using a Cyclone Phosphorimager (HP Company, Merident, CT). Relative mRNA expression level was ratio of targeted gene divided by CD31 gene. JNK kinase assays. HUVEC and mouse joints were lysed in lysis buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.1% Triton X-100, 0.75% Brij 96, 1 mM sodium orthovanadate, 1 mM sodium fluoride, 1 mM sodium pyrophosphate, 10 g/ml aprotinin, 10 g/ml leupeptin, 2 mM PMSF, 1 mM EDTA) as described [14]. JNK assay was performed as described [14] using GST-c-Jun (1-80) fusion protein as a substrate. We used 10 g GST-c-jun and 40 g total protein extract from EC lysates or the joint tissue in the in vitro kinase assays. Electrophoretic mobility shift assays (EMSA). The double-stranded oligonucleotide containing a B consensus site from the immunoglobulin- gene (Promega, Madison, WI) was used for EMSA. Preparation of nuclear extracts and EMSA were performed as described [14].

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Transfection and reporter assay. We used pBIIXLUC plasmid (B-LUC), which contains two B sites from the immunoglobulin- enhancer [14] and the JNK-dependent reporter gene system (FR-Luc and FA2-cJun) from Stratagene (PathDetect Reporting Systems; La Jolla, CA). Transfection of EC was performed using the DEAE-Dextran method [14]. HUVEC were transiently transfected with the VC or ES plasmids (1 g each) together with a promoter-reporter gene (1 g each). A constitutive expression vector for renilla luciferase (0.5 g each) was also transfected for normalization of transfection efficiency. Cells were left untreated or treated with TNF (10 ng/ml) for 12 hours. Luciferase activity followed by renilla activity was measured using a Berthold luminometer. All data were normalized as relative luciferase light units/renilla unit. Immunoprecipitation and immunoblot. Western blot analysis was performed as described [18]. HA-tagged endostatin was detected with antiHA (Roche Prognostics, Indiapolis, IN) and JNK1 protein was detected by antibody against JNK1 (Santa Cruz Biotechnology Inc.). The chemiluminescence was detected using an ECL kit according to the instructions of the manufacturer (Amersham, Arlington Heights, IL).

ACKNOWLEDGMENTS We thank George Kollias (Hellenic Pasteur Institute) for the TNF-transgenic mice; the Histology Core Facility at University of Rochester Medical Center for immunohistochemistry; and Bradford C. Berk (University of Rochester, NY) for helpful discussion and critical review of the manuscript. This work was supported by University of Rochester Starting Fund, Rochester Eye and Human Parts Bank, and a grant from NIH 1R01HL65978-01 to W.M. and AR4597-01 to E.M.S. RECEIVED FOR PUBLICATON DECEMBER 27, 2001; ACCEPTED MARCH 11, 2002.

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