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Abstract
Objective
Angiopoietin-1 (Ang1) is a potent angiogenic factor that can increase synovial angiogenesis and also en-hance osteoblast maturation and bone formation. However, its role in rheumatoid arthritis (RA) has not been well documented. Thus, we investigated roles of Ang1 in collagen-induced arthritis (CIA).
Methods
A recombinant adenovirus carrying the gene that encodes either cartilage oligomeric matrix protein (AdCOMP)-Ang1 (a modified form of Ang1) or LacZ (AdLacZ) was injected intravenously into CIA mice. Clinical, radiological, histopathological, and immuno-fluorescent analyses were performed. Serum levels of receptor activators of nuclear factor κ B ligand (RANKL) and osteoprotegerin (OPG) and expression of osteoblast maturation genes were analyzed.
Results
AdCOMP-Ang1-injected mice developed more severe inflammation than the AdLacZ-injected mice. However, there were no significant differences in cartilage damage and bone erosion. More PECAM-1-positive blood vessels were seen in the synovium of the AdCOMP-Ang1-injected mice than in those injected with AdLacZ. Interestingly, a lower number of TRAP-positive osteoclasts were observed in AdCOMP-Ang1-injected CIA mice than in the AdLacZ group when comparing sections obtained from joints showing similar synovial proliferation. The serum OPG/RANKL ratio and expression of osteoblast maturation genes, such as runt-related transcription factor 2, bone sialoprotein, type 1 collagen, osteopontin, and osterix, were significantly upregulated in the AdCOMP-Ang1 group.
Conclusion
COMP-Ang1 facilitates arthritis onset and increases synovial inflammation, but enhances osteoblast maturation, which in turn inhibits osteoclastogenesis by increasing the OPG/RANKL ratio in CIA. Our results sug-gest that careful investigation is necessary to delineate the possible therapeutic use of COMP-Ang1 as an adjunctive agent, in combination with antiinflammatory therapies, for the prevention of bone destruction in RA.
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Figure 1.
COMP-Ang1 increases erythema and joint swelling. (A) Representative photographs showing the gross features of the hind paws. The mouse injected with AdCOMP-Ang1 shows increased redness (right panel) compared with the Ad-LacZ mouse (left panel), despite showing comparable arthritic development in all paws. (B) The cumulative incidence of arthritis, (C) severity of arthritis, as assessed by a visual arthritis scoring system and (D) hind paw thickness were determined (n=10 for each group). Values are the mean± SEM, ∗p<0.05 vs. AdLacZ.
Figure 2.
AdCOMP-Ang1-injected mice show increased synovial proliferation and angiogenesis but no bone destruction. (A) Representative sections of the ankle joints stained with H&E (bars=250 μm), (B) mean pathological scores, and (C) mean radiological scores (n=10 for each group). Note the increased synovial proliferation (asterisk) in the joint of an AdCOMP-Ang1-injected mouse compared with PBS- and AdLacZ-injected mice. (D) Images showing PECAM-1+ blood vessels (bars=50 μm). (E) Density of blood vessels in the synovium (n=5-6 for each group). Note the increased angiogenesis in the synovium of an AdCOMP-Ang1-injected mouse compared with an AdLacZ. Values are the mean± SEM, ∗p<0.05 vs. AdLacZ.
Figure 3.
COMP-Ang1 decreases TRAP-positive osteoclasts. (A) Representative sections of the ankle joints stained with H&E (bars=500 μm) and TRAP (bars=50 μm) from an AdLacZ- or AdCOMP-Ang1-injected mouse with grade 3 synovial proliferation. The TRAP- stained image (lower panel) corres-ponds to the boxed area in the H&E-stained sections (upper panel). (B) Mean pathological scores and (C) total number of TRAP-positive cells in the joints showing grade 3 synovial proliferation from the AdLacZ- or AdCOMP-Ang1-injec-ted groups (n=5 for each group). Values are the mean± SEM, ∗p <0.05.
Figure 4.
COMP-Ang1 increases the OPG/RANKL ratio and the expression of the osteogenic genes. Ankle joint tissues were obtained from AdLacZ- and AdCOMP-Ang1-injected mice on day 45 (n=10 for each group). Real-time RT-PCR analysis for osteogenic and chondrogenic genes was performed. Values are the mean± SEM, ∗p<0.05, † p<0.01 vs. AdLacZ.
Table 1.
Sequences and accession numbers for the forward and reverse primers used in real-time RT-PCR
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