FIG. 5.

Immunohistochemical studies of ANK expression in the mouse growth plate and articular cartilage. (A) ANK is primarily expressed in the hypertrophic and calcification zones of the growth plate, and its expression is particularly strong in oxygen-rich areas of the growth plate undergoing vascular invasion. (B) ANK expression in the superficial zone of articular cartilage from 5-day postnatal mice corresponds to relatively high levels of oxygen from synovial fluid compared with the proliferative zone of articular cartilage where severe hypoxia results in very low ANK expression. (C) Immunohistochemical staining of ANK in meniscus shows high levels of expression; also note high levels of ANK in the superficial zone of the femoral condyle and tibial plateau in a mouse knee joint from a P7 animal. Autofluorescing anuclear RBCs in vessel walls emphasize high levels of ANK staining in these areas as well (white arrows). (D) H&E and (E) anti-ANK staining of human synovial tissue, showing high levels of expression of ANK in synoviocytes and in other regions of the tissue (F, fatty globules). (F) qRT-PCR showing very low levels of ANK expression in hypoxia in primary normal human articular chondrocytes. Values >1 indicate a relative increase in expression in hypoxia; values <1 indicate a relative decrease in expression in hypoxia. Data show that the expression of ANK is repressed in hypoxia, whereas the expression of VEGF is upregulated in hypoxia. *p < 0.05 for fold change in ANK expression in hypoxia vs. normoxia; n = 3. (G) qRT-PCR showing the relative expression of ANK in osteocytic MLO-A5 cells in normoxia and hypoxia. Note very high levels of ANK expression in normoxic conditions at times of mineralization (days 5–7) and repression of ANK expression in hypoxia at these same times. Results depict two separate experiments with qRT-PCR analyses performed in triplicate for each experiment.