Decreased angiogenesis and arthritic disease in rabbits treated with an αvβ3 antagonist (original) (raw)
The angiogenic growth factor bFGF enhances arthritic disease. To assess the effect of increased angiogenesis on arthritis development, the proangiogenic cytokine bFGF was added to antigen during arthritis induction. Recent evidence has shown bFGF to be upregulated and localized to the pannus–cartilage interface in RA tissues (5). The inclusion of bFGF in this model resulted in more consistent and accelerated arthritis than with antigen alone, characterized by greater joint swelling (Fig. 1a), increased pannus formation (Fig. 1b), and more frequent erosive disease (Fig. 1c). These results demonstrate that bFGF, an angiogenic growth factor, can exacerbate arthritis.
The proangiogenic cytokine bFGF intensifies arthritis. (a) The addition of bFGF during induction of AIA enhanced arthritis severity compared with OVA alone, with increased joint swelling, greater pannus formation (b), and earlier and more frequent erosive disease (c). Pannus development was graded on a relative scale 0–5 (0 = normal to 5 = macroscopic cartilage erosion) (ref. 23). All data are expressed as mean ± SE (n = 8). AIA, antigen-induced arthritis; bFGF, basic fibroblast growth factor; OVA, ovalbumin.
Integrin αvβ3 is selectively expressed on synovial blood vessels in AIA and in human RA. The rabbit AIA model resembles human RA in several important aspects, including histopathology and its response to therapeutic agents (18, 22). Rabbit AIA displays marked neovascularization accompanied by synovial growth and subsynovial inflammatory infiltrate (Fig. 2a). Notably, αvβ3 staining in AIA is restricted to synovial blood vessels (Fig. 2b), as in human RA, which is indicative of ongoing angiogenesis; blood vessels in sections from normal, nonarthritic synovium express little or no αvβ3 (data not shown). The earliest invasive blood vessels, although positive for αvβ3, do not stain for vWf (Fig 2b, arrows), reflecting the fact that vWf is a marker of differentiated endothelium (23). As vessels mature, αvβ3 is lost (24). The hyperproliferative synovial lining and subsynovial tissue also contain numerous fibroblasts and macrophages (Fig. 2a) (1). In contrast to cutaneous wound fibroblasts, which express integrin αvβ3 (25), little or no expression of this integrin was found on synovial fibroblasts (Fig. 2b), in agreement with previous studies (14, 15, 26, 27). With respect to synovial macrophages, αvβ3 expression was found to be restricted to osteoclasts and was not present in other monocyte/macrophage–derived cells (28).
AIA and human RA exhibit αvβ3 expression on angiogenic blood vessels. (a) Cryosections of infrapatellar tissue in AIA stained with H&E demonstrate marked neovascularization, synovial hypertrophy, and dense synovial inflammatory infiltrate relative to synovium from nonarthritic tissue (×100). (b) Immunofluorescent detection of blood vessels by vWf-specific antibody (green) and MAB LM609 directed to integrin αvβ3 (red) demonstrates colocalization of αvβ3 on the synovial endothelium (yellow merge) in rabbit AIA, as in human RA (×400). Note that microvascular angiogenic sprouts express only αvβ3 (arrows). H&E, hematoxylin and eosin; MAB, monoclonal antibody; RA, rheumatoid arthritis; vWf, von Willebrand factor.
Synovial neovascularization is inhibited by an αvβ3 antagonist that targets angiogenic blood vessels. Blockade of integrin αvβ3 by either antibody or peptide antagonists successfully inhibits neovascularization in cytokine and tumor models of angiogenesis in avian, murine, rabbit, and human tissues (11–13). To evaluate whether blockade of integrin αvβ3 could impact arthritis-associated angiogenesis, rabbits with AIA were treated intra-articularly 24 hours after arthritis onset and weekly thereafter for four weeks with the αvβ3 antagonist EMD 66203. This treatment resulted in a modest yet significant (P < 0.05) decrease in joint swelling compared with that in animals treated with the control peptide, EMD 69601 (Fig. 3a). Initial examination of knee joints revealed obvious periarticular vascularization in the control-treated animals (Fig. 3b). Cryosections of the infrapatellar fat-pad were examined with anti-vWf as a blood vessel marker. Tissues from rabbits treated with the αvβ3 integrin antagonist displayed considerably fewer blood vessels than tissues from control-treated rabbits (Fig. 4a). Blinded computer analysis of digital images revealed a 48% decrease in synovial neovascularization in αvβ3 antagonist–treated animals (Fig. 4b) (P < 0.01).
Joint swelling is reduced after αvβ3 antagonist administration in AIA. (a) Rabbits with AIA were treated with bilateral intra-articular αvβ3 antagonist (EMD 66203; 0.5 ml, 2 mg/ml) or control peptide (EMD 69601) beginning 24 h after arthritis onset, and weekly thereafter for 4 weeks (arrows). Joint swelling, defined as the increase in knee diameter from normal (nonarthritic), was significantly reduced by the αvβ3 antagonist (days 14–28, P < 0.05, ANOVA) compared with control peptide. (b) Periarticular vascularization (arrowheads) was prominent in control-treated AIA compared with antagonist-treated groups.
Synovial vascularity is decreased after αvβ3 antagonist treatment. (a) Cryosections of the infrapatellar tissue obtained 28 days after arthritis onset in animals treated with the αvβ3 antagonist or control peptide (days 1, 7, 14, and 21) were stained for vWf as a marker of blood vessels, detected with FITC-labeled secondary antibody, and were digitally imaged (×200). (b) The relative increase in area of fluorescent pixels per field vs. normal was computed to determine the angiogenic index as described in Methods. Synovial neovascularization was significantly inhibited by the αvβ3 antagonist (P < 0.050, Student's t test). Data are expressed as mean ± SE (n = 20).
To determine whether the αvβ3 antagonist was targeted to the vasculature in the arthritic synovium, we evaluated the distribution of a fluorescent-labeled form of this peptide, EMD 80838. Twenty-four hours after intra-articular injection, the fluorescent-conjugated αvβ3-binding peptide selectively localized to microvessels within the arthritic synovium, as determined by confocal microscopy (Fig. 5). These studies demonstrate that the αvβ3 antagonist targets synovial blood vessels in this model and inhibits angiogenesis in the arthritic synovium.
Fluorescent integrin antagonist colocalizes with synovial angiogenic microvessels. An FITC-conjugated αvβ3 antagonist peptide, EMD 80838 (cyclic-Arg-Gly-Asp-D-Phe-Lys-[fluoresceincarboxylic acid]; 0.5 mg/500 μl), was injected intra-articularly in rabbits with AIA. After 24 h, cryostat sections (5 μm) of synovial tissue were examined by confocal microscopy for the presence of peptide (green). Blood vessels were identified by antisera to vWF as detected with TRITC-labeled secondary antibody (red). Colocalization of αvβ3 antagonist is observed on angiogenic microvessels after signal merge (yellow), but not with mature vWF+ vessels (×630). TRITC, tetrarhodamine isothiocyanate.
Animals treated with the αvβ3 antagonist show reduced synovial infiltrate. To evaluate whether the observed angiogenesis inhibition in αvβ3 antagonist–treated animals was associated with a change in arthritis, several disease parameters were examined. Angiogenesis inhibition has been postulated to limit accessibility of the tissue to leukocytes (3); therefore, we first examined whether there was any decrease in the degree of synovial inflammatory infiltrate in animals treated with the antiangiogenic αvβ3 antagonist. Histological examination of the infrapatellar fat-pad revealed that treatment with the αvβ3 antagonist resulted in significantly fewer cells infiltrating the synovium (Fig. 6a). Computerized cell counting of digital images of these sections demonstrated a 39% reduction in synovial cellular infiltrate (Fig. 6b) (P < 0.05). Since only a small fraction of peripheral lymphocytes (1%–2%) express integrin αvβ3 (29), it is unlikely that the reduction in cellular infiltrate was due to leukocyte integrin inhibition. In fact, chemotaxis of AIA peripheral blood mononuclear cells (PBMCs) and polymorphonuclear neutrophils (PMNs) toward RA synovial fluid was not influenced by either the αvβ3-directed peptide or the control peptide on collagen (Fig. 6c) or fibronectin (data not shown). No leukopenia or other sign of hematologic or marrow toxicity was observed after treatment in either group (Table 1). Immunohistochemical analysis revealed no difference in the degree of colocalization between apoptotic cells and leukocytes, as determined by TUNEL and KEN-11 (anti–LFA-1) costaining studies between control and αvβ3 antagonist–treated rabbits (data not shown). Thus, the reduced synovial infiltrate observed in the αvβ3 antagonist–treated AIA synovium does not appear to result from direct effects of this compound on leukocyte number or migration.
Integrin αvβ3 antagonist reduces synovial inflammatory infiltrate in AIA without impairing leukocyte migration. (a) H&E stain of cryosections of the infrapatellar fat-pad 28 days after arthritis onset reveals a marked decrease in synovial cellular infiltrate in animals treated with the αvβ3 antagonist (days 1, 7, 14, and 21) compared with control. (b) Digital assessment of nuclei present in each field (three per joint, ×400) demonstrates that αvβ3 antagonist treatment resulted in a significant reduction in the cellular infiltrate (P < 0.05, Student's t test). Data are expressed as mean ± SE (n = 20). (c) In vitro chemotaxis on type II collagen toward synovial fluid by PBMCs or PMNs isolated from peripheral blood from rabbits with AIA. Data are expressed as mean ± SE of triplicate determinations. No effect of the αvβ3 antagonist on the migratory capacity of leukocytes was observed. PBMCs, peripheral blood mononuclear cells; PMNs, polymorphonuclear neutrophils.
No hematologic toxicity with αvβ3 antagonist treatment
The αvβ3 antagonist protects against erosive disease. Since pannus formation is thought to initiate progression to erosive disease (1), the effect of the αvβ3 antagonist on the development of pannus was examined. In control-treated animals, extensive pannus formation covering much of the femoral articular cartilage surface was observed, whereas treatment with the αvβ3 antagonist resulted in significant reduction of pannus development (P < 0.05) (Fig. 7a). The αvβ3 antagonist also significantly blocked the development of cartilage erosions. Gross erosions were present in 70% (14 of 20) of femoral condyles in control-treated AIA, yet only 20% (4 of 20) treated with the αvβ3 antagonist exhibited erosions (Fig. 7b) (P < 0.01).
Blockade of integrin αvβ3 decreases pannus formation and cartilage erosions. (a) Pannus development (arrowheads) was graded on a relative scale 0–5 (0 = normal to 5 = macroscopic erosion). Treatment with the αvβ3 antagonist significantly decreased pannus formation (P < 0.05, Student's t test) relative to control peptide. (b) Frontal sections of decalcified femoral condyle stained by H&E illustrate the protective effect of αvβ3 antagonist treatment on a representative sample of erosive disease (×10). (P indicates pannus, E indicates erosion, and the arrowhead indicates preservation of the articular cartilage in the antagonist-treated group.) Macroscopic cartilage erosions were decreased with αvβ3 antagonist treatment (P < 0.01, Student's t test). All data are expressed as mean ± SE (n = 20).
Vascular integrin αvβ3 is the preferred target in AIA. Recent studies suggest that both integrin αvβ3 and integrin αvβ5 can play a role in angiogenesis (30). Therefore, we compared the antiarthritic effects of the cyclic peptide with αvβ3 selectivity, EMD 66203, with that of a peptide with higher αvβ5-binding activity, EMD 85189. Notably, αvβ5 is also highly expressed on synovial fibroblasts and macrophages (14, 27). The peptide antagonist with greatest selectivity for αvβ3 demonstrated strongest inhibition of pannus development and erosive disease (Table 2), suggesting that αvβ3 was the primary therapeutic target in this model.
Integrin αvβ3 selective antagonist ameliorates AIA
Chronic arthritis is ameliorated by the αvβ3 antagonist. We extended these studies to evaluate the effect of αvβ3 blockade on well-established arthritis. AIA was allowed to progress for two weeks into the chronic phase (18) before the initiation of treatment. Despite the delay, joint swelling was significantly decreased by the αvβ3 antagonist (Fig. 8a). In this chronic model, treatment with the antagonist produced a significant antiangiogenic effect, with a 64% reduction in the angiogenic index compared with untreated AIA (P < 0.05) (Fig. 8b), and blockade of αvβ3 was again associated with a marked reduction in the synovial cell infiltrate (P < 0.05) (Fig. 8c). As observed previously with early administration during acute disease, delayed treatment with the αvβ3 antagonist decreased pannus formation and reduced the incidence of erosive disease compared with controls (Fig. 8, d and e). From these results, we conclude that angiogenesis contributes to disease severity as well as chronicity, and that an antagonist of integrin αvβ3 is an effective antiarthritic agent in well-established disease.
Chronic arthritis is ameliorated by an αvβ3 antagonist. (a) Administration of the antagonist (arrows) beginning 2 weeks after arthritis onset resulted in decreased joint swelling compared with controls (untreated) (P < 0.05, ANOVA). (b) Angiogenesis, as depicted by the angiogenic index (see Methods), was inhibited by αvβ3 antagonist treatment (P < 0.05, Student's t test). (c) Fewer infiltrating cells were observed in the synovium of αvβ3 antagonist–treated animals (P < 0.05, Student's t test), as assessed by digital computerized counting of nuclei. (d) Pannus was assessed as described in Methods. (e) A decrease in both pannus formation and cartilage erosion was observed in antagonist-treated animals relative to control. Data are expressed as mean ± SE (n = 12, antagonist; n = 10, control). HPF, high-power field; Tx, treatment.
Synovial vascular apoptosis is associated with αvβ3 antagonist treatment. Previous studies have demonstrated that antagonists of integrin αvβ3 induce apoptosis of angiogenic blood vessels (11–13). Therefore, we examined synovial tissues from the chronic arthritis model for the presence of apoptotic cells after treatment with the αvβ3 antagonist. Immunohistochemical detection of apoptosis within AIA synovium demonstrated increased (2.5-fold) TUNEL-positive vascular cells in the αvβ3 antagonist–treated animals (P < 0.01) (Fig. 9). These observations suggest that αvβ3 antagonists may inhibit angiogenesis in the arthritic synovium by inducing apoptosis of angiogenic blood vessels.
Vascular cell apoptosis is associated with αvβ3 antagonist treatment. (a)Synovium from rabbits in the chronic AIA model were stained with TUNEL immunostaining (red) as an indicator of apoptosis, (arrowheads)and anti-vWF (green) as a marker of blood vessels (×400). (b) Apoptosis associated with blood vessels was detected and quantified blindly as percent blood vessels containing apoptotic cells in 20 fields (×400) per specimen. Apoptosis was increased specifically in the vasculature of arthritic synovium after αvβ3 antagonist treatment (P < 0.01, Student's t test). TUNEL, terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling.