FIGURE 1.

NDNF expression is increased in the endothelium of muscle tissue after hind limb ischemic surgery. A, NDNF mRNA levels in non-ischemic and ischemic adductor muscle of WT mice on day 14 after hind limb ischemic surgery (n = 4), as measured by real-time PCR. NDNF mRNA levels were expressed relative to levels of 36B4 mRNA and normalized to non-ischemic muscle controls. B, NDNF protein expression in non-ischemic and ischemic skeletal muscle on day 14 after operation, as determined by Western blot analysis. Bottom panel, quantitative analysis of the NDNF protein levels relative to tubulin as evaluated by the ImageJ program. n = 4 in each group. C, plasma NDNF levels on day 14 after hind limb ischemic or sham (Control) operation, as determined by Western blot analysis. Bottom panel, quantitative analysis of plasma NDNF expression. n = 6 in each group. D, localization of NDNF in non-ischemic and ischemic limbs by immunohistochemical analysis. Tissue sections of non-ischemic and ischemic limbs were stained with an antibody against NDNF (red) and an antibody against CD31 (green), and merged images are shown. Scale bars = 50 μm. E, NDNF mRNA levels in whole non-ischemic muscle and endothelial cells isolated from non-ischemic or ischemic muscle tissue. n = 4 in each group. NDNF mRNA levels were expressed relative to levels of 36B4 mRNA and normalized to non-ischemic muscle controls. EC, endothelial cells. F, NDNF is secreted from cultured endothelial cells. HUVECs were cultured under conditions of normoxia (Norm) or hypoxia (Hypo) for 24 h. NDNF protein expression was determined in medium and cell lysates by Western blot analysis. G, NDNF mRNA levels in HUVECs treated with YC-1 (10 μm) or vehicle (dimethyl sulfoxide (DMSO)) under normoxic or hypoxic conditions as determined by real-time PCR. NDNF mRNA levels were expressed relative to levels of 36B4 mRNA and normalized to dimethyl sulfoxide controls under normoxic conditions. n = 4 in each group. H, NDNF mRNA levels in HUVECs after treatment with dimethylated glycine (DMOG) (100 μm), CoCl₂ (250 μm), or vehicle (Veh) for 6 h. NDNF mRNA levels were expressed relative to levels of 36B4 mRNA and normalized to vehicle controls. n = 4 in each group.