Adenovirus-based overexpression of tissue inhibitor of metalloproteinases 1 reduces tissue damage in the joints of tumor necrosis factor alpha transgenic mice.

OBJECTIVE: Rheumatoid arthritis is a prototype of a destructive inflammatory disease. Inflammation triggered by the overexpression of tumor necrosis factor alpha (TNFalpha) is a driving force of this disorder and mediates tissue destruction. Since matrix metalloproteinases (MMPs) are among the molecules activated by TNFalpha, we hypothesized that overexpression of their natural inhibitor, tissue inhibitor of metalloproteinases 1 (TIMP-1), in TNFalpha transgenic mice could inhibit the development of destructive arthritis. METHODS: Systemic treatment was carried out by replication-defective adenoviral vectors for TIMP-1, beta-galactosidase, or phosphate buffered saline (PBS), which were applied once at the onset of arthritis. Clinical, serologic, radiologic, and histologic outcomes were assessed 18 days after the treatment. RESULTS: The AdTIMP-1 group showed significantly reduced paw swelling and increased grip strength compared with the 2 control groups, whereas total body weight, TNFalpha, and interleukin-6 levels were similar in all 3 groups. Radiographic assessment revealed a significant reduction of joint destruction in the AdTIMP-1 group; this was confirmed by histologic analyses showing reduced formation of pannus and erosions in the AdTIMP-1 group compared with the AdLacZ and PBS control groups. The formation of arthritis-specific autoantibodies to heterogeneous nuclear RNP A2 was not observed in the AdTIMP-1 group but was present in the 2 control groups. CONCLUSION: These results indicate a central role of MMPs in TNFalpha-mediated tissue damage in vivo and a promising therapeutic role for TIMP-1.

Activation, differential localization, and regulation of the stress-activated protein kinases, extracellular signal-regulated kinase, c-JUN N-terminal kinase, and p38 mitogen-activated protein kinase, in synovial tissue and cells in rheumatoid arthritis.

OBJECTIVE: To investigate whether stress- and mitogen-activated protein kinases (SAPK/MAPK), such as extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK, are significantly activated in rheumatoid arthritis (RA) synovial tissue compared with their activation in degenerative joint disease; to assess the localization of SAPK/MAPK activation in rheumatoid synovial tissue; and to search for the factors leading to stress kinase activation in human synovial cells. METHODS: Immunoblotting and immunohistology by antibodies specific for the activated forms of SAPK/MAPK were performed on synovial tissue samples from patients with RA and osteoarthritis (OA). In addition, untreated and cytokine-treated human synovial cells were assessed for SAPK/MAPK activation and downstream signaling by various techniques. RESULTS: ERK, JNK, and p38 MAPK activation were almost exclusively found in synovial tissue from RA, but not OA, patients. ERK activation was localized around synovial microvessels, JNK activation was localized around and within mononuclear cell infiltrates, and p38 MAPK activation was observed in the synovial lining layer and in synovial endothelial cells. Tumor necrosis factor alpha, interleukin-1 (IL-1), and IL-6 were the major inducers of ERK, JNK, and p38 MAPK activation in cultured human synovial cells. CONCLUSION: Signaling through SAPK/MAPK pathways is a typical feature of chronic synovitis in RA, but not in degenerative joint disease. SAPK/MAPK signaling is found at distinct sites in the synovial tissue, is induced by proinflammatory cytokines, and could lead to the design of highly targeted therapies.

Determination of nifedipine and its three principal metabolites in plasma and urine by automated electron-capture capillary gas chromatography.

A sensitive, efficient, linear and reproducible capillary gas chromatographic method with electron-capture detection was developed for the quantitation of nifedipine and its primary metabolite M-I in plasma together with the urinary and principal metabolites M-II and M-III. On-column, rather than split-splitless, injection was employed to obviate oxidative degradation of nifedipine to M-I. The photosensitivity of nifedipine was re-examined under laboratory conditions and nifedipine was found to have a half-life in excess of two days when amber glassware and darkroom manipulations under red light were used. The method can determine nifedipine and its metabolites in plasma and urine after a single oral dose of 5 mg and can be applied to measure M-I production by human liver microsomes.

Human P450PCN1: sequence, chromosome localization, and direct evidence through cDNA expression that P450PCN1 is nifedipine oxidase.

P450PCN protein levels and nifedipine oxidase activities were quantitated in 12 human livers and were shown to be highly correlated. Antibody against rat P450PCN1 completely inhibited all nifedipine oxidase activity in three human liver samples. These results suggest that a human P450 related to rat P450PCN1 is the major form of P450 catalyzing nifedipine oxidation. The cDNA for a human P450, designated phPCN1, was isolated from a human liver lambda gt11 cDNA library, and sequenced completely. The deduced amino acid sequence is 77% similar to rat P450PCN1. By use of the adenovirus- and SV40-based expression vecotr p91023(B), the phP450PCN1 cDNA was expressed in COS cells and had high nifedipine oxidase activity, providing conclusive evidence that this P450 is the primary enzyme associated with metabolism and inactivation of this important drug. Using somatic cell hybrids, the P450PCN gene was localized to human chromosome 7.