Local gene delivery of heme oxygenase-1 by adeno-associated virus into osteoarthritic mouse joints exhibiting synovial oxidative stress S. Kyostio-Moore,

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Local gene delivery of heme oxygenase-1 by adeno-associated virus into osteoarthritic mouse joints exhibiting synovial oxidative stress S. Kyostio-Moore, D.S. Bangari, P. Ewing, B. Nambiar, P. Berthelette, C. Sookdeo, E. Hutto, N. Moran, J. Sullivan, G.L. Matthews, A. Scaria, D. Armentano Osteoarthritis and Cartilage Volume 21, Issue 2, Pages 358-367 (February 2013) DOI: 10.1016/j.joca.2012.11.002 Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Histopathological scoring of joints from STR/ort and CBA mice at 26–31 weeks of age. Scatter plot shows cartilage scores (H&E-stained sections) for individual joints (filled circles) and group median (horizontal line) (n = 8/group). Scoring was performed as described in Methods. Statistical analysis by Kruskal–Wallis showed no significant difference between the STR/ort groups while they were significantly different from CBA mice (P < 0.05). Osteoarthritis and Cartilage 2013 21, 358-367DOI: (10.1016/j.joca.2012.11.002) Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Localization of AAV transduction sites in mouse joints after IA delivery of various rAAV serotypes with LacZ. (A) Macroscopic analysis for localization of β-gal positive tissues in the mouse joints (sp, suprapatellar pouch) (B) Identification of β-gal positive cell types in each joint (n = 8/group). Bar graph indicates the number of joints positive for the cell type indicated. Analysis was performed 5 weeks after AAV administration using nuclear fast-stained joint sections. Osteoarthritis and Cartilage 2013 21, 358-367DOI: (10.1016/j.joca.2012.11.002) Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Histological analysis for localization of rAAV transduction in osteoarthritic STR/ort and normal CBA mouse joints. Top two panels: STR/ort joint infected with AAV1/LacZ. Images show entire joint (40×) and higher magnifications (400×) of skeletal muscle, synovium, adipose tissue, joint capsule and cartilage. Bottom two panels: STR/ort infected with AAV2/LacZ (inset shows synovial lining in suprapatellar region), AAV5/LacZ, AAV8/LacZ and AAV1/EV or CBA joint infected with AAV1/LacZ. Magnifications for each are shown (40× to 400×). Abbreviations: tp, tibial plateau; fc, femoral condyle; sy, synovium; sp, suprapatellar pouch; ad, adipose tissue: jc, joint capsule and ac, articular cartilage. Osteoarthritis and Cartilage 2013 21, 358-367DOI: (10.1016/j.joca.2012.11.002) Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Effect of oxidative stress on AAV transduction in primary synoviocytes in vitro. Primary rabbit synoviocytes were infected with AAV2/EGFP followed by 2 h H2O2-treatment as described in Methods and all endpoints were measured 3 days later. (A) AAV transduction by percentage of EGFP-positive cells and median fluorescence intensity (MFI) by FACS analysis. The levels represent mean ± 95% CI (n = 3/treatment; P values by Student's t test). (B) Effect of transient H2O2-treatment on intracellular ROS levels. Cells were treated with H2O2 for 2 h and ROS levels were determined using ROS sensitive fluorescent probe, APF. The histogram shows comparable increase in fluorescence intensity with 50 and 100 μΜ H2O2. All experiments were performed minimum of two times. Osteoarthritis and Cartilage 2013 21, 358-367DOI: (10.1016/j.joca.2012.11.002) Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 Localization of oxidative stress in mouse stiffle joints. (A) 3-NT immunostaining in osteoarthritic STR/ort (left panel) and normal CBA joints (right panel). Joint sections from 4 month-old mice were subjected to 3-NT IHC as described in Methods. Representative images (400× magnification) of 3-NT immunostaining (red) in the synovium, cartilage and periosteum are shown (synovium is indicated with an arrow; bn = bone; P = periosteum). (B) Quantitation of 3-NT immunostaining in the mouse joints. Each STR/ort (n = 7–8) and CBA (n = 3) joint was scored for the number of 3-NT positive cells as described in Methods. Scatter plot shows scores for individual joints (filled circles), group median (horizontal line) and P values (Mann–Whitney test). Osteoarthritis and Cartilage 2013 21, 358-367DOI: (10.1016/j.joca.2012.11.002) Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 Production and potency of HO-1 from an AAV1/HO-1 vector in vitro. 293 cells were infected with AAV1/HO-1. (A) HO-1 levels in conditioned media and cell lysates 3 days after infection by ELISA assay. (B) Effect of HO-1 production on intracellular ROS accumulation. The ability of HO-1 to reduce oxidative stress was determined using a ROS sensitive fluorescent probe, APF, added after transient H2O2-treatment 3 days post-AAV/HO-1 infection. The values represent mean ± 95% CI (n = 3–6/treatment; P values by Student's t test). All experiments were performed minimum of two times. Osteoarthritis and Cartilage 2013 21, 358-367DOI: (10.1016/j.joca.2012.11.002) Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions

Fig. 7 Cartilage evaluation after local delivery of AAV1/HO-1 into mouse OA joints. rAAV vectors were administered by IA route into mouse joints at 3–4 months of age and articular cartilage morphology was analyzed 3 months later. H&E-stained sections were scored for cartilage degradation, synovial inflammation and synovial proliferation. T. blue-stained sections were scored for cartilage proteoglycan content. Scatter plot (n = 8–12/group) shows cartilage scores for individual joints (filled circles) and group median (horizontal line) as described in Methods. Statistical analysis by Kruskal–Wallis showed no difference between the STR/ort groups while they were all different from CBA mice (P < 0.05). Osteoarthritis and Cartilage 2013 21, 358-367DOI: (10.1016/j.joca.2012.11.002) Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions

Fig. 8 Analysis of HO-1 expression in mouse synovial tissues 3 months after vector delivery. (A) HO-immunostaining for osteoarthritic STR/ort and normal CBA joints. Joint sections (n = 8–12/group) were stained as described in Methods. Representative HO-1 immunostaining (red) in the synovium of each group and isotype control antibody for STR/ort with AAV1/HO-1 are shown (matching joint). All isotype antibody controls were negative for growth plate staining while sections treated with HO-1 antibody stained endogenous HO-1 in the growth plate area (solid arrow; positive control for the HO-1 staining in all joints). (B) Scoring of synovial HO-1 immunostaining in the mouse joints. Scatter plot shows values for each STR/ort and CBA joint (n = 8–12/group) scored as described in Methods. Statistical analysis by Kruskal–Wallis showed no difference between the STR/ort treatment groups while both groups were different from CBA mice (P < 0.05). (C) AAV transgene expression from AAV1/LacZ in STR/ort mice 3 months after vector delivery. X-gal treated joints were sectioned and stained with nuclear fast (red) and location of β-gal activity (blue) was analyzed. Location of synovium is shown by arrows. Abbreviations: sy, synovium; fc, femoral condyle; tp, tibial plateau (magnification: top panel, 40×; bottom panel, 400×). Osteoarthritis and Cartilage 2013 21, 358-367DOI: (10.1016/j.joca.2012.11.002) Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions