The effect of rTMD proteins on angiogenesis in vitro and in vivo. (A) Growth-arrested HUVECs were treated with various Pichia-expressed rTMD proteins for 2 days, and then pulse labeled with BrdU for 2 hours. (B) Chemotaxis was assessed by Boyden chamber migration assay. EGF (10 ng/mL) was used as positive control. HUVECs were added in the upper compartment, and the bottom compartment was filled with various rTMD proteins. After 4 hours of incubation, HUVECs that migrated to lower face of the membrane were counted. HPF indicates high-power field. Values are the mean ± SD (n = 5), and similar results were obtained in at least 3 different experiments. *P < .05 and ***P < .001 compared with the control; #P < .05 and ###P < .001 compared with the rTMD123. (C) rTMD proteins show different angiogenic activity in vivo. Matrigel plugs containing equal molar concentrations of rTMD1, rTMD23, or rTMD123 were used in the murine angiogenesis assay. The amounts of hemoglobin indicated the angiogenic activity elicited by rTMD proteins. The data represent mean ± SD determined from 4-9 plugs. Similar results were obtained from at least 2 separate experiments; n represents the number of plugs for each group. **P < .01 compared with the PBS; #P < .05 compared with the rTMD123. (D) rTMD1 inhibited tube formation on Matrigel. HUVECs in the presence of 5% FBS and 3.75 μg/mL endothelial cell growth supplement were pretreated with various concentrations of rTMD1 for 30 minutes before being added to the Matrigel. After 8 hours of incubation, the tube length was measured using the MetaMorph program. The magnification bar is 100 μm. The data are the mean ± SD (n = 3). **P < .01 and ***P < .001 compared with the control. Similar results were obtained in at least 3 different experiments.