Figure 4

Effects of 2-methoxyestradiol and dimethyloxaloylglycine. Effects on synovial tissue, levels of hypoxia-inducible factor (HIF) 1α and viability of chondrocytes, and the effect of 2-methoxyestradiol (2ME2) on the expression of inflammatory cytokines. (a) to (d) Administration of 2ME2 (10 μM) or of dimethyloxaloylglycine (DMOG) (1 mM) did not induce inflammation or fibrosis of synovial tissue. A slight thickening of the synovial cell lining could be observed (b) 1 week following the repeated 2ME2 injections and (d) in osteoarthritic joints of STR/ORT mice. Immunostaining for CD45 revealed no relevant invasion of leukocytes into synovial tissue in (e) control joints, (f), (g) 2ME2-treated joints or (h) DMOG-treated joints. Large amounts of CD45-positive cells could be detected within bone marrow spaces. (i) Immunohistochemical detection of HIF-1α revealed strong staining in most articular chondrocytes of control joints of Balb/C mice. (j) 2ME2-treated joints showed a distinct reduction of HIF-1α-positive cells with less intense staining at 3 weeks. (k) Number of positive cells recovered in joints analyzed at 12 weeks. (l) DMOG treatment did not relevantly influence the number of positive cells. (m) Apoptosis in articular cartilage, visualized by the TUNEL method, was virtually absent in the control joints. (n) Treatment with 2ME2 resulted in a considerable number of TUNEL-positive cells, particularly in superficial layers at 3 weeks. (o) After 12 weeks, the number of positive cells declined but was still elevated compared with controls. (p) Only few cells were TUNEL-positive in the articular cartilage of DMOG-treated STR/ORT mice. Expression of (q) IL-1β and (r) IL-6 in cultured articular chondrocytes determined by quantitative RT-PCR under 21% or 1% oxygen and treatment with or without 2ME2. Treatment with the solvent dimethyl sulfoxide alone served as an additional control. Expression levels are shown as the mean ± standard deviation. *P <0.05. Bars = 100 μm.