Angiogenesis is an important phenomenon of synovial inflammation in RA . Following chronic inflammation, up-regulation of VEGF increases pathogenesis of RA, such as vascular permeability resulting in oedema, protein leakage, bone erosion and progressive destruction of the joints [26, 27]. More recent studies have addressed the role in arthritis of another important family of molecules involved in angiogenesis, namely the angiopoietins. These molecules, together with their cell-surface receptors Tie-1 and Tie-2, play a key role in the development of the vasculature. In RA, Ang-1 is expressed in human RA synovium in lining cells, macrophages, fibroblasts and endothelium [28, 29]. Like Tie-1, Tie-2 is also expressed on a variety of cells in the synovium and up-regulated in RA . Hence, the delicate balance between members of the Ang and Tie families may contribute to vascular formation in RA . Several other angiogenic growth factors, such as platelet-derived growth factor (PDGF), fibroblast growth factor (FGF)-2, epidermal growth factor (EGF), insulin-like growth factor (IGF-I), hepatocyte growth factor (HGF), TNF-α, transforming growth factor (TGF)-β, interleukin (IL)-1, IL-6, IL-8, IL-13, IL-15, IL-18, angiogenin, platelet-activating factor (PAF), angiopoietin, soluble adhesion molecules and endothelial mediator (endoglin), play an important role in angiogenesis in rheumatoid arthritis . The synovium of RA patients and joints from rats with adjuvant-induced arthritis contain increased amounts of FGF-2 . Rodent models have been used extensively to study the mechanisms underlying the VEGF-mediated angiogenic process in arthritic diseases and to develop new therapeutic interventions, including those based on inhibition of angiogenesis by targeting VEGF [15, 33, 34]. In our previous study we purified a CaMBP, NAP, which had angiogenic properties. The importance of calcium-binding proteins in angiogenesis and inflammation has also been reported earlier, proving that calcium-binding proteins are also potent angiogenic mediators [7, 35]. Earlier, our laboratory reported the proinflammatory role of CaMBPs isolated from ascites fluid from mouse mammary carcinoma cell lines that could activate respiratory burst . Consistent with previous reports, NAP isolated from SF of RA induces oedema in the footpad, revealing proinflammatory activity. Reports showing that the presence of CaMBPs at sites of acute and chronic inflammation have long been noted. Indeed, assessment of serum levels of CaMBP molecules have been suggested to track disease activity in patients with inflammatory disorders such as ulcerative colitis, chronic inflammatory bowel disease, psoriatic arthritis (sPA) and RA , and is also a valuable marker [36-38]. We have developed a model using NAP similar to the AIA model of RA in Wistar rats to examine the role of NAP in the development of this disease. Our results show that the levels of NAP and VEGF in AIA and NIA animals were found to increase in serum. Similar to other reports [36, 39, 40], NAP levels in the serum elevated gradually after the onset of arthritis, with the highest level at 21 days after induction. Treatment with antibodies such as anti-TNF-α antibody has influenced the expression of other proinflammatory cytokines involved in RA . Antibodies against calcium- and membrane-binding protein have reduced the accumulation of neutrophils in air pouch models of acute gouty arthritis . Annexins are another class of CaMBPs which induce angiogenesis via stimulation of VEGF production. S100A4 induce angiogenesis through interaction with annexin II on the surface of endothelial cells . Treatment with anti-S100A12 antibodies, anti-renal cell carcinoma antigen (RAGE) antibodies and soluble-RAGE (sRAGE) and CaMBPs have reduced inflammation effectively in animal models of arthritis . Consistent with previous reports, our data demonstrate that treatment with anti-NAP mAb of AIA or NIA rat models effectively reduces paw swelling, degree of redness and flexibility of the rear ankle joints, indicating the neutralization and potential therapeutic effect of these antibodies. Quantification of growth factor VEGF and NAP by ELISA indicated an increased amount of VEGF or NAP correlating with the progression of the disease, whereas in the case of anti-NAP mAb-treated animals, a decrease in the amount of NAP or VEGF levels in sera was evident. The effect of anti-NAP mAb on proliferation of endothelial cells is especially visible when observing blood vessel formation in synovium. Histopathological studies showed clearly the inhibition of blood vessel formation in H&E staining. Consistent with the above results, immunohistochemical staining for CD31, Flt1 or VEGF revealed reduced synovial vessels, thus suggesting that anti-NAP mAb also influences angiogenesis in synovium. Our results have shown that there was extensive neovascularization in synovium of NIA or AIA rats due to VEGF or NAP. As there is inhibition of revascularization and reduction in VEGF or NAP levels in serum, anti-NAP mAb is affecting the angiogenesis either directly or indirectly. Additionally, these results confirm that NAP is a proinflammatory/pro-arthritic factor, as well as being a pro-angiogenic factor.
In conclusion, the present data indicate that NAP is a potent proinflammatory and pro-angiogenic factor in NIA or AIA rat models. Anti-NAP mAb treatment decreased significantly the severity of arthritis and improved the histological findings in established NIA or AIA rat models. Anti-NAP mAb also reduced the neovascularization and proinflammatory proteins, resulting in a decrease in MVD and thereby an anti-arthritic effect. Anti-inflammatory and anti-angiogenic effects are likely to be interdependent mechanisms, resulting in a profound anti-arthritic effect in NIA or AIA rat models. Anti-NAP mAb can also be used as a diagnostic tool for detection of NAP in sera and effusions of patients with inflammatory disorders. These findings, showing that in-vivo administration of anti-NAP mAb suppressed arthritis on established AIA or NIA rats, suggest that anti-NAP mAb treatment may serve as a new and additional therapeutic modality for RA. However, research needs to be continued to understand the importance of NAP, and further clinical trials using anti-NAP mAb may prove to be much more effective and cost-effective, and with fewer side effects.