Rheumatoid arthritis (RA) is a chronic inflammatory disease associated with intense angiogenesis (1). Formation of new blood vessels (angiogenesis) is one of the earliest histopathologic findings in RA and appears to play a critical role in the generation and prolonged survival of the hypercellular synovial membrane and invasive pannus in RA (2, 3). First, neovascularization increases the nutrient supply to tumorlike synovium. Second, the expanded endothelial cell surface area in an inflamed joint maximizes the routes of ingress that allow immune and inflammatory cells to adhere and migrate into the synovium. Finally, activated endothelial cells provide a potent source of proinflammatory cytokines, chemokines, and growth factors (4). Therefore, activated endothelium and angiogenesis should be good targets for therapeutic intervention in RA. Reduced inflammation arising from the diminished angiogenesis could further ameliorate new blood vessel formation, because many mediators of inflammation are, themselves, proangiogenic.
Although no therapy specifically targeting angiogenesis in RA is currently available, many existing treatments for RA have been shown to possess some degree of antiangiogenic activity. For example, methotrexate, a common treatment in patients with RA, was shown to inhibit endothelial cell proliferation in vitro (5). Anti–tumor necrosis factor α (anti-TNFα) antibody treatment significantly decreased serum levels of vascular endothelial growth factor (VEGF) and reduced vascularity in RA joints (6, 7). In addition, AGM-1470 (a low molecular weight synthetic analog of fumagillin), D-penicillamine, paclitaxel, gold-containing compounds, sulfasalazine, cyclosporine, and glucocorticoids also exhibit angiostatic properties (8–12). More recently, several antiangiogenic agents targeting VEGF and αvβ3 integrins have been shown to be effective in attenuating arthritis in animal models (13–17).
Tie2 is an endothelium-specific receptor tyrosine kinase that is required for embryonic vascular development (18). Tie2 also plays a critical role in pathologic angiogenesis and disease development (19–25). Several studies have demonstrated the presence of both Tie2 and its ligands, angiopoietin 1 (Ang-1) and Ang-2, in RA synovium (26–31). Recently, we showed that levels of Tie2 and Ang-1 were elevated in human RA synovium, and that blocking Tie2 activation using a soluble Tie2 receptor (ExTek) inhibited arthritis-induced angiogenesis in a synovium vascular window model (32). The current study was designed to determine whether neutralization of Tie2 has a therapeutic benefit in arthritis development. Our results show that systemic delivery of a Tie2 inhibitor using a gene therapy approach delayed disease onset, reduced disease severity, and protected against bone erosion in a collagen-induced arthritis (CIA) mouse model. Thus, our study has identified a new target for arthritis treatment.
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- MATERIALS AND METHODS
Angiogenesis is an essential step in the development of arthritis. In this study, we demonstrate that systemic delivery of a Tie2 inhibitor (ExTek) using a gene therapy approach (AdExTek) significantly inhibited disease onset, as well as the incidence and severity of arthritis in a CIA model. AdExTek treatment was able to inhibit arthritis progression even after initiation of the disease. Reduced disease development and progression correlated with a significant reduction of neovascularization in arthritic synovium, suggesting that inhibition of the disease is secondary to inhibited angiogenesis. Most strikingly, we show that neutralization of Tie2 protects against bone destruction in the CIA model, possibly through inhibition of RANKL production, which reveals a significant clinical benefit of targeting this signaling pathway in arthritis therapy.
Angiogenesis is a multistep process that requires sequential activation of different growth factors and their cognate receptors. Of the molecular mechanisms identified to date, activation of endothelial receptor tyrosine kinases (RTKs) by polypeptide growth factors appears to play a pivotal role in blood vessel growth and differentiation (41, 42). Importantly, RTKs for 2 families of angiogenic growth factors, the VEGF receptor (VEGFR) family and the Tie2 family, are expressed predominantly on vascular endothelial cells, making them attractive targets for antiangiogenic therapy (41, 42). VEGF and its receptors are present in arthritic synovium, and neutralization of VEGF function inhibited arthritis development in animal models (13, 16, 17). In a previous study, we observed significantly elevated levels of Tie2 and its ligand, Ang-1, in human RA synovium (32). Consistent with our report, several studies have also shown the presence of Tie2 and Ang-1 in arthritic synovium (26–29). We further demonstrated, using a synovium vascular window model, that blocking Tie2 action with AdExTek significantly inhibited angiogenesis in arthritis (32). Results of our current study further illustrate the therapeutic potential of targeting the Tie2 signaling pathway in arthritis treatment.
Results of genetics studies indicate that the Tie2 and VEGFR pathways work in a coordinate and collaborative manner in regulating angiogenesis, with VEGFR acting during the early stages of vessel development and Tie2 acting later to promote angiogenic remodeling as well as vessel maturation and stabilization (41, 42). We have shown that by blocking Tie2 function using ExTek, adult angiogenesis and tumor angiogenesis were inhibited, even in the presence of VEGF (20, 22). Neutralization of either the Tie2 pathway or the VEGFR pathway for a mammary tumor could significantly block tumor angiogenesis and tumor growth (20, 22). These results suggest that the Tie2 and VEGFR pathways are 2 independent mechanisms essential for tumor angiogenesis. This observation was further confirmed by another study of a human melanoma, A375v (24). The ability of AdExTek to inhibit angiogenesis despite the presence of VEGFR suggests that disrupting the angiogenic program at stages distal to endothelial activation will provide effective and perhaps more globally useful inhibitors of pathologic neovascularization. In the setting of arthritis development, it would be of great interest to compare the effects of blocking both Tie2 and VEGFR signaling with those of blocking the signaling of either one of them.
The significant inhibitory effect of AdExTek on arthritis supports the notion that angiogenesis is an essential step in arthritis development and progression. Indeed, we observed robust angiogenesis at a very early stage, days before appearance of the clinical signs of disease onset (Figure 2), which is consistent with a report that angiogenesis starts very early and may even antedate the specific clinical and histologic signs of inflammation in human RA (43). Furthermore, the therapeutic effects were more profound when the treatment was given before disease onset rather than after its initiation, which implies that it might be easier to inhibit vascular formation than to induce vascular regression after the establishment of matured vessels. Antiangiogenic therapy requires long-term delivery of inhibitors in vivo. Although the adenoviral vector is very efficient in transducing genes in vivo, transgene expression ceases after 1 week to 10 days, due to host immune surveillance. It appeared that ExTek expression correlated with inhibition of the disease. The significant differences in disease development and progression between the treated group and the control group diminished at a later time. With the fast development of better viral vectors that are much less immunogenic or not immunogenic at all, this limitation should be solved in the near future.
Bone destruction in later stages of arthritis is one of the major reasons for the progressive disability and morbidity in patients with RA (39). Surprisingly, we observed significant protection against bone damage in the AdExTek-treated joints compared with controls, even when the mice showed identical clinical signs. This protection most likely results from reduced angiogenesis, which evidently would limit inflammatory cell infiltration. Reduced angiogenesis also means a limited nutrient supply and a reduction in the levels of inflammatory cytokines and growth factors that could be either transported by the blood vessels or directly produced by the vascular endothelium.
The molecular mechanism responsible for bone destruction in RA is less clear. Several lines of evidence support a role for osteoclasts in the pathogenesis of bone erosion in RA (29). Therefore, we examined the effects of ExTek on osteoclasts in joint tissues. However, we did not observe any significant difference in the number of osteoclasts between the control and ExTek-treated groups, indicating that targeting Tie2 action does not affect the production or recruitment of osteoclasts. Additionally, we measured RANKL levels, an important mediator of bone destruction, in grade 3 joint tissues from both groups. Interestingly, we observed a significant decrease in RANKL levels in ExTek-treated joint tissues compared with those from controls. These findings imply that blockade of Tie2 activation by ExTek inhibited RANKL expression and thus might contribute to bone protection. TNFα plays a central role in RA, not only in inflammation but also in bone destruction. TNFα-transgenic mice develop a spontaneous and destructive polyarthritis with rapidly progressive destruction of bone, which mimics human RA in many aspects (44). Anti-TNFα therapies not only effectively block synovial inflammation but also affect tissue destruction (7, 45). Results of our previous study showed that TNFα up-regulates Tie2 signaling in multiple ways. Collectively, these findings indicate that TNFα might regulate RANKL production in endothelial cells via Tie2 activation.
Quantitative radiographic scoring of RA in humans has been in use for decades, providing unique information that is not available by clinical examination (46). However, there is a lack of similar approaches in animal studies, especially in mouse models. In this study, we adapted a method of phase-contrast radiography, which is a new class of plain radiography based on the shifts of x-rays as they pass through an object (47). Conventional radiography is based on the absorption of x-rays rather than on their displacement. A phase-contrast radiograph contains all of the absorption information of a conventional radiograph plus the additional information related to x-ray shifts (evident predominantly at the edges of objects). It offers very high resolution and a digital image. Recent advancements indicated that phase-contrast radiography might have a role in arthritis imaging in the near future (37, 40, 48). Because of the availability of different transgenic mouse strains for arthritis studies, this technique should be very valuable.
In conclusion, this study clearly shows the clinical, histologic, and radiologic benefits of specific Tie2 blockade on disease onset, progression, and bone destruction in the CIA mouse model. These findings identify a new molecular target for arthritis treatment. It opens up new possibilities for RA treatment strategies and warrants clinical trials of specific Tie2 inhibitors for the treatment of arthritis.