Arthritis & Rheumatism, Volume 63,
November 2011 Abstract Supplement
Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Chicago, Illinois November 4-9, 2011.
Heme Oxygenase-1 Lentiviral Transduction of Human Osteoarthritic Osteoblasts Down-Regulates Inflammatory, Degradative and Senescence Markers.
Guillen1, Maria Isabel, Clerigues1, Victoria, Castejon2, Miguel Angel, Gomar3, Francisco, Alcaraz1, Maria Jose
In previous works we demonstrated that the antioxidant enzyme heme oxygenase-1 (HO-1) protects against inflammatory and degenerative effects of IL-1b in osteoarthritic (OA) chondrocytes and synoviocytes. Osteoblasts play an important role in remodelling processes in OA. In this work we studied HO-1 effects on inflammatory, degradative and senescence markers in OA osteoblasts stimulated with IL-1b. Overexpression of HO-1 was performed by infection of these cells with lentiviral vectors.
The knee specimens were obtained from 46 patients with the diagnosis of advanced OA (71.5±7.1 years, mean ± S.E.M.) undergoing total knee joint replacement. The pieces of trabecular bone were obtained from the femoral condyles and tibial plateau, and cells were isolated after collagenase digestion. Osteoblasts were cultured in osteogenic medium and used at third passage. Lentiviral vector stocks were generated in HEK293T cells by calcium phosphate-mediated transient transfection of psPAX2, pMD2G and pWXL plasmids for 24 and 48h. The titers of lentiviral stocks were in the range of 35 × 105 IU/ml as determined by immunocytochemical analysis of HEK293T-infected cells. Osteoblasts culture was infected with LV-HO-1 flag or control empty vector, LV(-) for 24 h. After infection and culture in osteogenic medium for 2 days, osteoblasts were stimulated with IL-1b (10 ng/ml) for 24 h. HO-1 expression was measured by immunofluorescence using an anti-flag monoclonal antibody. PGE2 was evaluated by RIA, IL-6, TNF-a, IL-10, matrix metalloproteinases (MMPs) and transcription factors NF-kB and AP-1 by ELISA, COX-2, mPGES-1, osteocalcin (OCN), osteopontin (OPN), collagen 1A1, collagen 1A2, osteoprotegerin (OPG), RANKL, and senescence markers (hTERT, caveolin and p21) by real-time PCR.
Lentiviral vectors showed not toxicity on OA osteoblasts, evaluated by trypan blue exclusion test. The results showed that IL-b decreased HO-1 expression in cells treated with IL-1b and transfected with LV(-). However, in LV-HO-1 flag-transfected cells, IL-b did not alter the expression of HO-1. These effects were observed at protein and mRNA levels. IL-1b significantly increased the production of PGE2, COX-2, mPGES-1, IL-6, TNF-a, MMP- 1, 2 and 3, with respect to basal conditions. In LV-HO-1 flag cells, all these mediators were significantly reduced. In contrast, HO-1 overexpression increased the production of IL-10, OCN, OPN, collagen 1A1, and the ratio OPG/RANKL, with respect to IL-1b control cells. The evaluation of senescence parameters showed that in cells transfected with LV-HO-1 flag hTERT was enhanced and caveolin decreased. The analysis of transcription factors showed HO-1 overexpression significantly reduced NF-kB activation in osteoblasts stimulated with IL-1b.
In this study we have demonstrated that HO-1 down-regulates the inflammatory response and cellular senescence besides exerting a positive effect on osteoblastic markers. HO-1 may be a novel therapeutic target in inflammatory and degradative processes in OA bone
To cite this abstract, please use the following information:
Guillen, Maria Isabel, Clerigues, Victoria, Castejon, Miguel Angel, Gomar, Francisco, Alcaraz, Maria Jose; Heme Oxygenase-1 Lentiviral Transduction of Human Osteoarthritic Osteoblasts Down-Regulates Inflammatory, Degradative and Senescence Markers. [abstract]. Arthritis Rheum 2011;63 Suppl 10 :1782