Rheumatoid arthritis (RA) is a multi-systemic autoimmune disease of unknown etiology that is characterized by hyperplastic synovial membrane capable of destroying adjacent articular cartilage and bone.  The characteristics of rheumatoid arthritis (RA) pathology include the infiltration of inflammatory leukocytes, the proliferation of synovial cells, and the presence of extensive angiogenesis, referred to as rheumatoid pannus. A critical phenomenon occurring in the early stages of synovial inflammation is angiogenesis, which commences with the activation of endothelial cells by a variety of stimuli, including pro-inflammatory cytokines and growth factors such as vascular endothelial growth factor (VEGF). The affected endothelial cells (ECs) then begin to digest the basement membrane, proliferate, migrate and eventually differentiate to form a tubular structure.

1. Synovial tissues were minced into 2 to 3 mm pieces, and treated for 4 hours with 4 mg/ml of type I collagenase in primary cell culture system at 37°C in a 5% CO₂ atmosphere.

2. Dissociated cells were then resuspended in primary cell culture system, supplemented with 10% FCS, 2 mM glutamine, penicillin and streptomycin, and then plated in 75 cm2 flasks.

3. After overnight culturing, the non-adherent cells were removed and adherent cells cultivated in primary cell culture system plus 10% FCS.

4. Cultures were kept at 37°C in a 5% CO₂ atmosphere, and the medium was replaced every 3 days.

5. At confluence, the cells were passed by diluting 1:3 with fresh medium and re-cultured until used.

6. Synoviocytes, from passages 4 through 8, were used for each experiment.

7. RA fibroblast-like synoviocytes (FLSs) were washed in primary cell culture system and then incubated for an additional 24 hours in serum-free primary cell culture system supplemented with insulin-transferrin-selenium A.

8. The cells (3 × 10⁴ cells/well) were seeded in triplicate into 24-well plates (Nunc, Roskilde, Denmark) in serum-free primary cell culture system with transforming growth factor (TGF)-β.

9. After the indicated hours of incubation, cell viability was tested by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and the cell-free media were collected to measure VEGF165 concentration in the culture supernatants.

10. Synovial fluid was obtained with informed consent from RA patients with joint effusion, and stored at -20°C in a refrigerator.

11. All samples were obtained according to the guidelines approved by the Ethics Committee.

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