Inflammation is normally a protective response to pathogenic, traumatic, or toxic injury, reducing tissue damage and leading to resolution or repair (1). In chronic inflammatory diseases such as rheumatoid arthritis (RA), persistent inflammation itself causes tissue damage (2). The mechanisms by which inflammation persists rather than resolves remain incompletely understood, particularly as in RA, where a persisting injurious agent has not been definitively identified. Chronic inflammatory diseases are a major health problem worldwide, leading to chronic pain and disability (3–5). Identifying the factors that lead to persistent inflammation should lead to novel therapies, thus reducing the burden of chronic inflammatory diseases by facilitating resolution or repair.
Angiogenesis is tightly regulated during normal growth, wound healing, and the female reproductive cycle (6), whereas persistent angiogenesis is characteristic of chronic inflammatory disease. For example, blood vessel growth within the inflamed synovium is typical of both early and established RA (7–9). In chronic synovitis, a shift in the balance from antiangiogenic to angiogenic factors promotes endothelial cell (EC) proliferation and blood vessel growth (10). Chronic inflammation maintains blood vessel growth by the secretion of angiogenic factors by macrophages and other cells (11) while synovial angiogenesis can further facilitate inflammation by increasing plasma extravasation and enhancing inflammatory cell recruitment (12, 13).
The fibroblast growth factor (FGF) system plays an important role in angiogenesis and in the maintenance of vascular integrity (14, 15). FGF-2 promotes EC proliferation and the physical organization of ECs into tube-like structures. It also stimulates the proliferation of fibroblasts that give rise to granulation tissue in wound healing (16). FGF-2–induced angiogenesis occurs in the absence of inflammation (17, 18), a characteristic which distinguishes it from many other angiogenic factors such as vascular endothelial growth factor.
The 2 methionine aminopeptidase (MetAP) isoforms, MetAP-1 and MetAP-2, are cotranslational regulators of protein synthesis. MetAP remove the initiator methionine from growing polypeptide chains, a prerequisite for a variety of biologic processes such as protein stability (19, 20). Inhibition of MetAP-2 prevents the selective removal of the N-terminal methionine and potently inhibits EC proliferation, a prominent mechanism in angiogenesis. PPI-2458 is an antiangiogenic fumagillin analog that is an orally active, irreversible inhibitor of MetAP-2 via covalent modification of His-231 in the catalytic site of the enzyme. This triggers growth arrest of ECs in the late G1 phase of the cell cycle, inhibiting EC proliferation and angiogenesis without affecting inflammatory cytokine release (19, 21, 22) or inducing apoptosis in ECs (23). PPI-2458 has reduced toxicity and greater selectivity for angiogenesis inhibition versus inflammation as compared with another fumagillin analog, AGM-1470/TNP-470 (23, 24).
PPI-2458 has been shown to reduce synovitis and bone and cartilage damage in collagen-induced arthritis in mice and peptidoglycan–polysaccharide–induced arthritis in rats (21, 25). Those studies and studies of other antiangiogenic strategies (20, 26–29) indicate that angiogenesis inhibition may have therapeutic potential in RA in humans. However, treatment of chronic inflammatory diseases with sustained inhibition of angiogenesis may lead to adverse events due to the blockade of physiologic blood vessel growth. An antiangiogenic strategy targeted to key phases in the development of chronic inflammation may have a more favorable risk/benefit ratio.
Intraarticular injection of low doses (0.03%) of carrageenan induces resolving synovitis without angiogenesis in rats, whereas synovitis persists when it is accompanied by synovial angiogenesis (30). This led us to hypothesize that angiogenesis during the early phase of synovitis may be a key factor determining its persistence and, where this is the case, that brief treatment with an angiogenesis inhibitor at the onset of arthritis could prevent its subsequent persistence. We stimulated angiogenesis by intraarticular injection of FGF-2 in rats. Low-dose carrageenan alone did not cause angiogenesis, whereas combined injection of carrageenan and FGF-2 was used to model combined inflammation and angiogenesis, as seen in early synovitis in humans. We used this model to evaluate the possible contribution of angiogenesis to the transition from acute to persistent synovitis.
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In the present study, we examined whether angiogenesis may be a key factor in the transition from acute to persistent inflammation in an animal model of synovitis. We have shown that at a dose of 0.03%, carrageenan alone did not induce angiogenesis. Injection of carrageenan alone was associated with a transient synovitis that resolved after 14 days. When angiogenesis was stimulated with 6 pmoles of FGF-2 at the time that synovitis was induced by intraarticular injection of carrageenan, the synovitis persisted for at least 4 weeks. Inhibition of this effect of FGF-2 by PPI-2458 suggests that this chronicity is mediated by angiogenesis rather than by other actions of FGF-2.
The mechanisms leading to persistent inflammation in human diseases remain unclear. Autoimmunity contributes to inflammation and tissue damage in diseases such as RA, although its contribution to persistence in early RA remains incompletely understood. Evidence of autoimmunity, such as circulating anti–citrullinated peptide antibodies, has limited sensitivity in predicting persistence in early synovitis, and immunosuppression does not prevent persistence in the majority of patients. Many other chronic inflammatory diseases (e.g., psoriasis and inflammatory bowel disease) display little evidence of autoimmunity. Angiogenesis inhibition may reduce arthritis severity in animal models of autoimmunity (21, 23, 25, 27, 28, 37, 38). Our data indicate that angiogenesis inhibition may prevent the progression from acute to chronic inflammation in situations where new blood vessel growth is the key switch to persistence.
In the present study, we used FGF-2 and PPI-2458 and specific outcome measures of angiogenesis and inflammation to determine the effects of angiogenesis inhibition on the persistence of inflammation. FGFs are implicated in the initiation and development of synovial hyperplasia and inflammation in RA (39). FGF-2 is up-regulated at the cartilage–pannus interface in RA (40), and increased synovial fluid levels of FGF-2 are associated with greater disease severity (41, 42). FGF-2 was also shown to augment antigen-induced arthritis in rabbits (27), and FGF-2 overexpression worsened inflammation and joint damage in adjuvant-induced arthritis in rats (43).
Our data support previous evidence that FGF-2 is proangiogenic but not directly proinflammatory (44–46). FGF-2 is mitogenic for ECs and acts via specific FGF-2 receptors displayed on these cells. In some experimental systems, FGF-2 can additionally enhance vascular integrity (14) and increase adhesion molecule expression by cytokine-activated ECs, thereby synergistically enhancing the recruitment of inflammatory cells (18). However, we found that a single intraarticular injection of FGF-2, either alone or together with carrageenan, was not sufficient to increase joint swelling or macrophage infiltration at 24 hours, despite its ability to increase EC proliferation. The FGF-2–induced increase in chronic synovitis was inhibited both by the brief administration of PPI-2458 in our study and by repeated administration of an antiangiogenic inhibitor of αvβ3 integrin in antigen-induced arthritis in a previous study (27). FGF-2, therefore, may contribute to arthritis by increasing synovial angiogenesis, rather than through any direct effect on inflammation.
PPI-2458 inhibits human EC proliferation by inhibiting MetAP-2. It does not reduce angiogenic growth factor production (19, 21, 22) or induce apoptosis in ECs (23). However, high doses of PPI-2458 may have effects in addition to angiogenesis inhibition, and mediation of its therapeutic effects by angiogenesis inhibition has been uncertain in previous studies (23). We found that PPI-2458 administration abolished synovial angiogenesis prior to reduction in synovitis, whereas synovitis reduction followed the discontinuation of PPI-2458. This indicates that PPI-2458 may exert its effects not through the direct inhibition of inflammation, but rather through the inhibition of angiogenesis.
Synovial angiogenesis is an early feature of RA, and occurs very early in the development of persistent synovitis in animal models (30, 37). Synovial angiogenesis is also a feature of chronic synovitis in both humans and animals (30, 47, 48). In the present study, we demonstrated increased EC proliferation during chronic synovitis 29 days after coinjection of carrageenan and FGF-2. It is likely that angiogenesis on day 29 was mediated by factors other than the injected FGF-2, since FGF-2 alone induced only a transient increase in EC proliferation that had normalized after 5 days. Angiogenesis during chronic synovitis may be mediated by an imbalance between a variety of angiogenic and antiangiogenic factors (12). Further studies are needed to determine which molecules sustain synovial angiogenesis in human or animal models.
Mechanisms by which acute synovitis may lead to chronic arthritis remain incompletely understood. Clinical trials aiming to induce remission in early synovitis by intensive immunosuppression have raised the prospect that it may eventually be possible to modify the course of, for example, RA, without the need for lifelong medication. Immunosuppression alone, however, has not yet achieved this goal, and other factors may be important in the transition from acute to chronic synovitis. Angiogenesis is a feature of early, as well as persistent, synovitis in humans (7–9), and our findings indicate that synovial angiogenesis may be a factor leading to persistent synovitis. Synovitis typically progresses more rapidly in animal models than does arthritis in humans, and further work is needed to determine the duration of any window of opportunity in early synovitis during which inhibition of angiogenesis may facilitate its resolution. Furthermore, since there may be multiple factors that lead to persistence, angiogenesis inhibition may have greatest therapeutic potential in combination with other pharmacologic agents. Similar combined approaches with angiogenesis inhibitors have proven successful in oncology (49, 50).
Sustained inhibition of angiogenesis that is begun during the induction, or at the onset, of synovitis can reduce inflammation and joint damage (21, 23, 27, 37, 38). However, adverse effects of long-term angiogenesis inhibition on physiologic blood vessel growth may outweigh the therapeutic benefit in chronic inflammatory diseases. Our data indicate that short-term angiogenesis inhibition targeted to a key period in the development of inflammation has the potential to achieve sustained clinical benefit. Clinical studies are needed to determine whether brief antiangiogenic treatments can facilitate resolution in early synovitis, at the onset of synovitis in a previously uninvolved joint, or at reactivation of previously quiescent inflammation. A greater understanding of the mechanisms underlying persistence in different clinical scenarios will be necessary in order to realize this potential.
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All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Ms Ashraf had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design.Ashraf, Mapp, Walsh.
Acquisition of data.Ashraf, Mapp, Walsh.
Analysis and interpretation of data.Ashraf, Mapp, Walsh.