Copper chelation with tetrathiomolybdate suppresses adjuvant-induced arthritis and inflammation-associated cachexia in rats

Abstract

Tetrathiomolybdate (TM), a drug developed for Wilson's disease, produces an anti-angiogenic and anti-inflammatory effect by reducing systemic copper levels. TM therapy has proved effective in inhibiting the growth of tumors in animal tumor models and in cancer patients. We have hypothesized that TM may be used for the therapy of rheumatoid arthritis and have examined the efficacy of TM on adjuvant-induced arthritis in the rat, which is a model of acute inflammatory arthritis and inflammatory cachexia. TM delayed the onset of and suppressed the severity of clinical arthritis on both paw volume and the arthritis score. Histological examination demonstrated that TM significantly reduces the synovial hyperplasia and inflammatory cell invasion in joint tissues. Interestingly, TM can inhibit the expression of vascular endothelial growth factor in serum synovial tissues, especially in endothelial cells and macrophages. Moreover, the extent of pannus formation, which leads to bone destruction, is correlated with the content of vascular endothelial growth factor in the serum. There was no mortality in TM-treated rat abnormalities. TM also suppressed inflammatory cachexia. We suggest that copper deficiency induced by TM is a potent approach both to inhibit the progression of rheumatoid arthritis with minimal adverse effects and to improve the well-being of rheumatoid arthritis patients.

Figure 1

Suppression of clinical arthritis by tetrathiomolybdate (TM) treatment in adjuvant-induced arthritis rats. TM was orally administered daily to female Lewis rats, from 2 weeks before immunization to day 17. TM-treated rats, 10 mg/kg/day (n = 8); control rats (n = 8). (a) Arthritis score, (b) paw volume, and (c) mean body weight were assessed every second day after onset of arthritis.

Figure 2

Morphological features and histopathological aspects of the hindlimb in AIA rats. (a) Control rats and (b) rats treated with tetrathiomolybdate (TM). Joint swelling, redness, and edema of the foot in AIA was clearly reduced with TM administration at day 17 after immunization. Histopathological studies using hematoxylin and eosin staining of the foot joint also revealed (c) a marked decrease of synovial inflammatory cell infiltrate and synovial lining hyperplasia compared with (d) control rats. (c), (d) Original magnification × 40. AIA, adjuvant-induced arthritis; MC, monocyte; SL, synovial lining cell; OC, osteoclast.

Figure 3

Histopathological scores of the hindlimb in AIA rats fed with TM or deionized water. Mononuclear cell infiltration, pannus invasion into the cartilage and bone, and vascularity were measured by microscopic examination scores of the sections on two separate occasions (see Histological examination). We measured the scores of 16 hindlimbs (both hindlimbs of each rat). AIA, adjuvant-induced arthritis; TM, tetrathiomolybdate.

Figure 4

Immnunostaining for VEGF in synovial tissue in AIA rats. (a),(c), (e) AIA rats treated with deionized water and (b),(d) tetrathiomolybdate (TM). Immunohistochemical staining was performed with the Vecto Stain avidin–biotin peroxidase complex kit (Vector Laboratories, Burlingame, CA, USA). Synovial tissues sections were stained with (a)–(d) mouse anti-VEGF monoclonal IgG antibodies (1:200 dilution, in PBS; Santa Cruz Biotechnology, Santa Cruz California, USA) and (e) a normal mouse IgG (1:200 dilution, in PBS). Positive immunostaining was indicated by brownish deposits. The counterstain was an aqueous solution of hematoxylin. (a), (b) Original magnification × 40; (c)–(e) original magnification × 400. AIA, adjuvant-induced arthritis; ET, endothelial cell; SL, synovial lining cell.

Figure 5

VEGF levels in the serum are correlated with the extent of arthritis in rats. (a) VEGF levels in the serum are higher in control rats than in tetrathiomolybdate (TM)-treated rats (* P < 0.01). VEGF was analyzed by ELISA, as described in Materials and methods. Data represent the mean ± standard deviation. (b) A significant positive relationship between VEGF levels in the serum and the severity of pannus formation in hindlimbs of adjuvant-induced arthritis rats, which includes both the control group and the TM-treated group. VEGF, vascular endothelial growth factor.