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Effect of Direct Angiogenesis Inhibition in Rheumatoid Arthritis Using a Soluble Vascular Endothelial Growth Factor Receptor

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Effect of Direct Angiogenesis Inhibition in Rheumatoid Arthritis Using a Soluble Vascular Endothelial Growth Factor Receptor Effect of Direct Angiogenesis Inhibition in Rheumatoid Arthritis Using a Soluble Vascular Endothelial Growth Factor Receptor 1 Chimeric Protein TOMOHISA SEKIMOTO, KOICHI HAMADA, YUICHI OIKE, TOMOMI MATSUOKA, MASANORI MATSUMOTO, ETSUO CHOSA, TOSHIO SUDA, NAOYA TAJIMA, and KEN-ICHI YAMAMURA ABSTRACT. Objective. We evaluated the effect of direct angiogenesis inhibition in synovium of patients with rheumatoid arthritis (RA), using a soluble vascular endothelial growth factor receptor 1 (VEGFR1) chimeric protein. Methods. Dispased cells from active RA synovial tissues were cocultured on OP9 stromal cells. Control synovial tissues were obtained from patients with injury of the anterior cruciate ligament. Chimeric protein (30 µg/ml) of the extracellular domain of VEGFR1 fused to the Fc portion of human IgG1 (VEGFR1-Fc) was added to culture medium. After 10 days, the cells were stained with anti-CD31 antibody and anti-Tie-2 antibody. Results. Endothelial cells from patients with active RA had high angiogenic growth capacity compared with controls. Proliferation of these endothelial cells was strongly suppressed by VEGFR1-Fc. Quantitative analysis revealed that VEGFR1-Fc inhibited angiogenesis in a dose dependent manner. Conclusion. VEGFR1-Fc is able to suppress angiogenesis in rheumatoid synovium, suggesting that direct inhibition of angiogenesis activity could serve as a novel therapeutic strategy to prevent progressive synovial hyperplasia and inflammatory reactions in active RA. (J Rheumatol 2002;29:240–5) Key Indexing Terms: ANGIOGENESIS INHIBITION ANGIOGENIC GROWTH CAPACITY CHIMERIC PROTEIN VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTOR 1 RHEUMATOID SYNOVIUM Rheumatoid arthritis (RA) is a chronic systemic inflamma- tion, bone erosion, and subsequent joint deformities is the tory disease of unknown etiology. RA occurs worldwide in hallmark of the disease. No therapeutic management all ethnic groups. The characteristic feature of RA is hyper- method presently in use is curative. The course of RA is plasia of synovial cells with angiogenesis, although there are quite variable, and difficult to predict in an individual a variety of systemic manifestations. The potential of the patient. Some patients experience relentless progressive persistent synovial inflammation to cause cartilage destruc- polyarthritis with marked joint destruction. An elevated degree of capillary congestion is seen in the proliferation of inflamed synovial tissue in active RA. From the Department of Orthopedic Surgery, Miyazaki Medical College, Miyazaki; and the Division of Developmental Genetics, Institute of Recently, it has been reported that angiogenesis precedes all Molecular Embryology and Genetics, Kumamoto University School of other features in early RA, on the basis of biopsy samples Medicine, Kumamoto, Japan. from the synovia of patients with monoarthritis who later Supported by a grant from Tanimura Hospital, Nobeoka, Miyazaki, 1 Japan. developed typical RA . Therefore, increased angiogenesis in T. Sekimoto, MD, PhD, Department of Orthopedic Surgery, Miyazaki rheumatoid synovial tissues could serve as the primary Medical College, and Division of Developmental Genetics, Institute of target of therapeutic strategy. Molecular Embryology and Genetics, Kumamoto University School of Several angiogenic factors are likely to be important in Medicine; K. Hamada, PhD; Y. Oike, MD, PhD; T. Suda, MD, PhD, Division of Cell Differentiation, Institute of Molecular Embryology and neovascularization. Several positive regulators of angiogen- Genetics, Kumamoto University School of Medicine; T. Matsuoka, MD; esis have been identified, e.g., the fibroblast growth factor M. Matsumoto, MD; E. Chosa, MD, PhD; N. Tajima, MD, PhD, family, the transforming growth factor family, hepatocyte Department of Orthopedic Surgery, Miyazaki Medical College; K. α Yamamura, MD, PhD, Division of Developmental Genetics, Institute of growth factor, tumor necrosis factor (TNF- ), vascular Molecular Embryology and Genetics, Kumamoto University School of endothelial growth factor (VEGF), angiogenin, interleukin 8 Medicine. (IL-8), and angiopoietins2. Negative regulators include Dr. Sekimoto and Dr. Hamada made an equal contribution to this paper. thrombospondin3, the 16 kilodalton fragment of prolactin4, Address reprint requests to Dr. T. Sekimoto, Department of Orthopedic Surgery, Miyazaki Medical College, 6500 Kihara, Kiyotake, Miyazaki the secreted protein that is acidic and rich in cysteine 5 6 7 889-1692, Japan. E-mail: [email protected] (SPARC) , angiostatin , and endostatin . The interplay Submitted February 20, 2001; revision accepted August 13, 2001. between positive and negative regulators of angiogenesis is Personal non-commercial use only. The Journal of Rheumatology Copyright © 2002. All rights reserved. 240 The Journal of Rheumatology 2002; 29:2 Downloaded on September 29, 2021 from www.jrheum.org thought to give rise to the complex patterns of vasculariza- clonal; Pharmingen, San Diego, CA, USA) or control Mab, and washed in tion observed in different tissues and organs8. PBS 2 times. They were incubated with a 1/500 dilution of anti-mouse The endothelial cell-specific mitogen VEGF is an impor- biotinylated antibody in 3% BSA/PBS, washed twice in PBS, incubated with avidin/biotinylated horseradish peroxidase complex (Vector tant mediator of physiological and pathological angiogen- Laboratories, Burlingame, CA, USA) for 30 min, and again washed in PBS esis9. VEGF is essential for embryonic development, and twice. They were then stained with diaminobenzidine tetrahydrochloride loss of even one VEGF allele results in embryonic substrate for 5 min, rinsed in tap water for 2 min, and mounted. The lethality10,11. VEGF is also required for growth and survival sections were photographed under an Olympus AX70 microscope. during early postnatal life12. However, in the fully devel- Production of recombinant fusion protein. As described20, a recombinant oped animal, VEGF is likely to be involved mainly in active fusion gene containing the extracellular domain of VEGFR1 and the Fc portion of human IgG1 was constructed. VEGFR1-Fc fusion proteins, angiogenesis processes such as corpus luteum develop- produced by COS7 cells transfected with the fusion gene in serum-free ment12. VEGF binds to its tyrosine kinase receptors conditioned medium23, were purified over protein-A columns (Affi-Gel VEGFR1 (Flt-1) and VEGFR2 (Flk-1/KDR), both of which protein A, Bio-Rad, Hercules, CA, USA). The purity and disulfide linked are expressed on endothelial cells10. Mice homozygous dimerization of the soluble VEGFR1 chimeric protein were assessed by negative for either receptor die between embryonic days 8.5 Coomassie brilliant blue staining of sodium dodecyl sulfate-polyacry- lamide gels. and 9.513,14. Strong experimental evidence links VEGFR2 9 In vitro culture of endothelial cells from synovial tissues. The stromal cell activation to VEGF induced mitogenesis and angiogenesis . line OP924 was maintained in α-modified minimum essential medium (α- Recent studies have unexpectedly shown that VEGFR1 may MEM, Gibco BRL, Gaithersburg, MD, USA) supplemented with 20% fetal function in angiogenesis mainly as a ligand-binding mole- calf serum (FCS; JRH Bioscience, Lenexa, KS, USA). Synovial tissues cule, rather than as a signaling tyrosine kinase15. were incubated with 0.01% collagenase (Sigma, St. Louis, MO, USA) in α- Several investigations have shown that concentration of MEM for 20 min at 37°C. The dispased cells were drawn through a 23G needle, deposited into a polystyrene tube, and centrifuged at 500 g for 10 VEGF in synovial fluid is significantly higher in patients min. Debris and aggregated cells were removed with a nylon mesh. The with RA than in controls, and that VEGF is strongly cells in suspension were counted, and viability was tested according to expressed in rheumatoid synovial lining cells and subsyn- Trypan blue exclusion criteria. These cells (3 × 104 cells/well) were ovial macrophages16-18. Further, serum concentration of cultured on OP9 stromal cells in RPMI-1640 (Gibco BRL) with 10% FCS and 10–5 M 2ME (Sigma) at 37°C in humidified 5% CO air, in the pres- VEGF is higher in patients with RA than in controls or 2 19 ence or absence of VEGFR1-Fc chimeric protein. After 10 days in culture, patients with other arthritides . Thus, there is speculation endothelial cells were stained using an anti-CD31 antibody (WM-59, that VEGF is involved in the development of inflammation mouse anti-human monoclonal), an anti-Tie-2 antibody (C9C3, mouse anti- and tumor-like proliferation of synovial tissue, and may be human monoclonal)25, and a Vector M.O.M. Immunodetection Kit (Vector important in the disease activity of RA. Laboratories, Burlingame, CA, USA). It has been reported that the experi- 26,27 We examined inhibition of angiogenesis in patients with mental agents in this study do not directly damage cells in vitro . After CD31 immunohistochemical staining, the number of CD31 positive cells RA, using a soluble VEGFR1 chimeric protein in which the under different culture conditions was counted per well. The average N-terminal extracellular domain of Flt-1 has been fused to number of 4 wells of the tissue culture from each subject was represented. an Fc fragment of human IgG120. The results suggest that Cultures were done in replicate. suppression of angiogenesis may be beneficial in the treat- Statistical analysis. All values were expressed as the mean ± standard devi- ment of active RA. To
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