Volume 11 Issue 6 - November 20, 2009 PDF
Amelioration of collagen-induced arthritis in rats by adenovirus-mediated PTEN gene transfer
Chrong-Reen Wang1, Ai-Li Shiau2, Chao-Liang Wu3,*

1 Section of Rheumatology, Department of Internal Medicine, 2 Departments of Microbiology and Immunology, 3 Department of Biochemistry and Molecular Biology, National Cheng Kung University, Tainan, Taiwan

Arthritis and Rheumatism 58: 1650–1656 (Jun 2008)

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FIGURE 1. Characterization and bioactivity of PTEN expressed by AdPTEN. A, Detections of PTEN, pAkt, and total Akt in RASFs treated with AdPTEN or AdLacZ in the presence of TNF-α by immunoblot analysis. Ratios between the intensity of the bands corresponding to pAkt and Akt, as determined by densitometric analysis, were shown. B, Detection of PTEN-HA transgene expression in the ankle joints of CIA rats administered intraarticularly with AdPTEN. C, Effect of AdPTEN on the migration (n=5) (C) and tube formation (n=3-4) (D) of endothelial cells. **= p < 0.01 versus AdGFP or mock control. Effect of AdPTEN on VEGF production by RASFs in the presence of EGF (E) or bFGF (F) from five different RA patients (n=3). p < 0.0001 for AdPTEN versus AdLacZ.
The phosphatidylinositol 3-kinase (PI3-kinase)/Akt pathway is known to be activated in rheumatoid arthritis (RA) synovial tissue, which impacts cell growth, proliferation, survival, and migration. Phosphatase and tensin homolog deleted from chromosome 10 (PTEN) functions as a negative regulator of PI 3-kinase signaling, thus blocking Akt activation. The aim of this study was to examine the effect of PTEN gene transfer in rats with collagen-induced arthritis (CIA). Expressions of PTEN transgene in RASFs infected with adenoviral vector encoded human PTEN cDNA (AdPTEN) were verified by immunoblot analysis. Reduced pAkt/Akt levels in AdPTEN-infected RASFs were also noted. Furthermore, AdPTEN inhibited the migration and tube formation of human endothelial cells. These results confirmed the biological activities of PTEN expressed by AdPTEN. RASFs are one of the significant sources of VEGF in the synovial fluid. Since growth factors, such as EGF and FGF, present in the synovial fluid of RA patients can upregulate VEGF expression in these cells, we examined whether AdPTEN reduced VEGF production by RASFs in the presence of growth factors. VEGF produced from AdPTEN-treated RASFs was significantly lower than that from AdLacZ-treated cells in the presence of either EGF or bFGF. AdPTEN dose-responsively reduced VEGF production in four of five RASFs (Figure 1).

To assess whether PTEN gene transfer reduced Akt phosphorylation in vivo, expressions of pAkt and total Akt in the ankle homogenates of individual rats were examined at different time points. In the CIA rats treated with either AdLacZ or PBS, the levels of Akt activation, which were expressed as the ratio of pAkt/Akt, were elevated over time, in particular on days 14 and 16. However, reduction of Akt activation was evident on day 10 in AdPTEN-treated rats. Although their pAkt/Akt ratios were also increased with time, Akt phosphorylation was at a much lower level compared with that in their control counterparts. Our results indicate down-regulation of Akt phosphorylation in CIA rats. The articular index and ankle circumference in the PTEN-treated CIA rats were significantly smaller than those in their control counterparts, indicating that PTEN gene transfer resulted in a significant reduction of joint inflammation. On day 17, the radiologic score, based on joint space width, degree of bony destruction, and soft tissue swelling, was also significantly lower in AdPTEN-treated rats. Histopathologic examination of the joint tissues from AdLacZ-treated ankles revealed synovial hyperplasia, cartilage erosion, and inflammatory infiltrates with leukocytes. However, in the AdPTEN-treated ankles, joint tissues displayed relatively mild changes with no erosion on cartilages. Furthermore, the histologic score of synovial hyperplasia, cartilage erosion, and leukocyte infiltration was significantly lower in the AdPTEN-treated rats compared with their control counterparts (Figure 2).
FIGURE 2. Levels of Akt activation in the ankle joints and clinical parameters of arthritis in rats immunized with collagen on days 0 and 7 followed by treatment with AdPTEN, AdLacZ or PBS on day 8. A, Changes in the protein levels of pAkt and total Akt during the time course of CIA. Ratios between the intensity of the bands corresponding to pAkt and Akt, pAkt and β-actin, as well as Akt and β-actin were calculated. The fold increase in the ratios shown below the blots is the ratio in each lane relative to the ratio obtained from the control lane from the sample collected on day 7 from CIA rats. Except for the control lane which represents a pooled sample from three animals due to inadequate amounts of samples, each lane represents a sample from one animal. Three animals at each time point were examined. AdPTEN reduced the articular index score (B) and ankle circumference (C) from day 13 onward (n = 20, ***=p < 0.001), as well as the radiologic scores of the ankle joints on day 17 (n=6, *= p < 0.05) (D).

There were fewer microvessels in the synovium from AdPTEN-treated rats than that from AdLacZ-treated rats. Quantitative data revealed reduced microvessel density in the AdPTEN-treated synovium. TUNEL assay also showed enhanced apoptosis in their synovium. There was a twofold increase in the number of apoptotic cells in AdPTEN-treated samples compared with their control counterparts (Figure 3). The levels of VEGF and IL-1β were significantly lower in the AdPTEN-treated ankles than those in their AdLacZ-treated counterparts. However, the level of TNF-α did not differ between the rats treated with AdPTEN and AdLacZ. Whereas the level of MMP-2 was unaffected by AdPTEN treatment, the production of MMP-9 was significantly decreased in the AdPTEN-treated ankles in comparison with their AdLacZ-treated counterparts, as determined by gelatin zymography.
FIGURE 3. Histopathological and immunohistochemical analyses of joint tissues on day 17 from arthritic rats treated with AdPTEN or AdLacZ. A, Representative AdLacZ-treated joint sections show synovial hyperplasia, cartilage erosion, and inflammatory infiltrates with leukocytes. AdPTEN-treated joint sections reveal relatively mild changes with no erosion on cartilages (original magnification × 40). B, The histologic scores of ankle joints (n=6, **= p < 0.005). C, Representative joint sections from CIA rats treated with AdPTEN reveal fewer microvessels in the synovium compared with their AdLacZ-treated counterparts, as identified by staining for von Willebrand factor. The lower two panels show the magnified images (× 100) of the area indicated by the boxes in the upper panels (original magnification × 40). D, Microvessel density in the synovium determined by averaging the number of von Willebrand factor-positive microvessels in three fields (at × 100 magnification) of the highest vessel density in each section (n=6, **= p < 0.005). E, In situ apoptosis in the synovium assessed by the TUNEL assay (original magnification × 400). F, TUNEL-positive cells were counted from three fields (at × 400 magnification) of the highest density of positive-stained cells in each section (n=3, *= p < 0.05).

This is the first in vivo study to demonstrate that intraarticular gene therapy with adenoviral vectors encoding PTEN can ameliorate arthritic symptoms in the rat CIA model mimicking the joint inflammation seen in human RA. The effectiveness of PTEN gene delivery opens the possibility of using this approach for the treatment of RA. Furthermore, our results also strongly implicate PI3K/Akt pathway as a therapeutic target for the treatment of RA or other inflammatory diseases.
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