Effects of TIMP-1 and TIMP-3 gene transfer on invasiveness, proliferation and apoptosis of rheumatoid arthritis (RA) synovial fibroblasts (RA-SF)

Abstract

TIMPs play a key role in counter balancing the action of MMPs and have been associated with cell proliferation, inhibition of angiogenesis and induction of apoptosis. Here, we investigated the effects of TIMP-1 and TIMP-3 gene transfer on cartilage invasion, proliferation and apoptosis of RA-SF. RA-SF were transduced with an adenoviral vector expressing human TIMP-1 (AdTIMP-1) or TIMP-3 (AdTIMP-3). Transduction efficacy was assessed by LacZ staining of RA-SF that were transduced with an adenoviral β-galactosidase construct. Untransduced and mock transduced RA-SF were used as controls. TIMP-1 was measured by ELISA in the culture supernatants of AdTIMP-1 transduced and mock transduced cells every 10 days until 60 days after transduction. Proliferation was assessed by 3H-thymidine incorporation, and the rate of spontaneous apoptosis as well as FasL induced cell death was determined by a histon fragmentation assay. AdTIMP-1 and AdTIMP-3 transduced RA-SF and control RA-SF were co-implanted with human articular cartilage under the renal capsule of SCID mice for 60 days and their invasiveness was evaluated on paraffin sections using a semiquantitative score. Transduction efficacy was 67%, and TIMP-1 levels in the supernatants of AdTIMP-1 transduced cells were 51.5 ± 6.5 μg/ml as compared to 8.7 ± 3.4 μg/ml in the mock transduced cells. These levels of TIMP expression were maintained for at least 60 days. AdTIMP-1 and AdTIMP-3 gene transfer resulted in an inhibition of proliferation (35% and 40% vs. mock, respectively; P < 0.05). Transduction of RA-SF with AdTIMP-3 but not TIMP-1 increased spontaneous apoptosis (+24%; vs. mock, P < 0.05) as well as susceptibility to FasL-induced cell death (+23% vs. mock, P < 0.05). In the SCID mouse model, untransduced and mock transduced RA.SF deeply invaded the cartilage (scores: 2.5 ± 0.2 and 3.2 respectively). In the AdTIMP-1 and AdTIMP-3 transduced RA-SF, invasion was inhibited clearly (scores 0.9 ± 0.4 and 1.2 ± 0.2 respectively) Both AdTIMP-1 and AdTIMP-3 gene transfer inhibit proliferation of RA-SF and reduce cartilage invasion. In contrast to TIMP-1, adenoviral gene transfer with TIMP-3. has a strong pro-apoptotic effect on RA-SF and facilitates Fas mediated cell death. These results indicate that gene transfer of TIMPs may be a useful approach to inhibit joint destruction in RA.