(A) mRNA and protein levels of DKK1 and DKK2 at the same time points (0.5, 8, and 12 hours) during morphogenesis in Matrigel were measured by RT-PCR (left) and Western blotting (right). (B) Human DKK1 or DKK2 promoter activities were measured by luciferase reporter assay. HUVECs cultured on gelatin-coated plates were transfected and transferred to gelatin-coated plates (gelatin) or Matrigel-coated plates (morphogenesis) 6 hours later. Luciferase reporter activity was measured 16 hours later. Data represent mean ± SD. (C) HUVECs stably expressing eGFP plus control (CTL) shRNA, eGFP plus DKK1 shRNA, or DKK1 plus control shRNA were established with lentivirus. Stable transfectants were plated on Matrigel-coated plates at a density of 1.5 × 10⁵ cells/well and incubated for 18 hours. Capillary-like networks, which completely differentiated into tube-like structure, were quantified with Image-Pro Plus software. (D) Proliferative indices of HUVECs transfected with eGFP or DKK1 were assessed by [³H]-thymidine incorporation assay. (E) HUVECs stably expressing eGFP, DKK2, control shRNA, or DKK2 shRNA. Morphogenesis of the transfectants on Matrigel was analyzed as described in C. (F) Proliferative indices of HUVECs transfected with eGFP or DKK2 were accessed as described in D. Data represent mean ± SD. **P < 0.01; ***P < 0.001.

(A–F) Isolectin B4 staining of whole-mounted P4 retinas of WT or DKK2 Tg mice (A and D). White broken lines indicate the range of vessel extension (A). Scale bars: 500 μm. White dots indicate filopodia extended from tip cells (D). Scale bars: 100 μm. Quantification of vessel densities (B), sprouting length (C), tip cell number (E), and filopodia (F). (G–I) Isolectin B4 staining of whole-mounted P12 retinas (G). Scale bars: 100 μm. Quantification of vessel density in the ganglion (first) (H) and the outer nuclear (third) cell layer (I). (J–M) Aortic segments were harvested from WT and DKK2 Tg mice (n = 7 per group). (J) Endothelial sprouts forming branching cords from the margins of aortic segments were photographed with a phase microscope. Scale bars: 200 μm. (L) Dynamic movement of endothelial sprouts from the margins of aortic segments was captured as real-time video (see Supplemental Video 1). Scale bars: 40 μm. Arrows indicate filopodia. Sprouting scores (K) and quantification of tip cell numbers (M). Sprouting scores were scored from 0 (least positive) to 5 (most positive). Data represent mean ± SD. *P < 0.05; ***P < 0.001. Box indicates 25%~75% value and whisker indicates media value in box plot (B, C, E, F, H, and I).

(A) HUVECs stably expressing eGFP, DKK1, or DKK2 were analyzed for Cdc42 activities. (B) Stable transfectants were cultured on Matrigel-coated plate (morphogenesis) for 2 hours and Cdc42 activities were measured. (C and D) HUVECs were transiently transfected with expression plasmids encoding eGFP control or eGFP dominant negative mutant of Cdc42 (Cdc42 N17). After 24 hours, these cells were plated on Matrigel-coated plates and incubated with PBS or DKK2 (1.5 μg/ml) for 2 hours. Then microphotographs were taken (C) and filopodia number was quantified (D). Arrowheads indicate filopodial extension. Data represent mean ± SD. **P < 0.01. Scale bars: 20 μm. (E) HUVECs stably expressing eGFP or DKK2 were incubated with sFRP (200 ng/ml) for 24 hours and Cdc42 activity was measured.

(A and B) Matrigel plugs treated with VEGF (200 ng) and DKK2 (1 μg) were excised from mice 5 days after injection (n = 5 per group). Scale bars: 100 μm. CD31 staining (A) and quantification of hemoglobin concentrations in a Matrigel plug (B). White arrows indicate CD31-stained vessels. (C–F) Corneal angiogenic responses induced by VEGF-containing (200 ng) or DKK2-containing (1 μg) micropellets. Scale bars: 500 μm. Asterisks indicate micropellet inserted (C). 5 days after pellet implantation, Evans blue was injected i.v. After 30 minutes, each mouse eye was photographed. Scale bars: 200 μm (D). CD31 and NG-2 staining of cryosections of the eye (E). Scale bars: 100 μm. Green, CD31 positive; red, NG-2 positive; blue, DAPI. Pericyte coverage calculated as a ratio of the NG-2 to CD31 staining area (F). Data represent mean ± SD. ***P < 0.001.

(A and B) Intramuscular injection of DKK2 increased blood perfusion and reduced the probability of necrosis in the ischemic hind limb in mice. Tissue perfusion rate (%/min) was defined as the fraction of blood exchanged per minute in the vascular volume by time-series analysis of indocyanine green dye. Blood perfusion rate of the hind limb was measured at postoperative day 0 (POD 0). Correlation between regional perfusion rates of the ischemic hind limbs at POD 0 and limb necrosis levels at POD 3 was determined. The x axis shows the regional perfusion rate of the ischemic hind limbs (poor perfusion rate: 0%~30%/min; moderate perfusion rate: 30%~100%/min; good perfusion rate: 100 < %/min). Normal hind limbs typically demonstrate a perfusion rate above 400%/min. (C–J) Increase of myocardial repair after DKK2 injection. Myocardial injection of DKK2 decreased the LV infarct size as assessed by TTC staining at 1 week after MI (C and D). Representative images taken from a Masson’s trichrome–stained section (muscle is stained red, collagen is stained blue) (E and F). Microvessel staining with CD31 (G) and the TUNEL assay (I) on cardiac muscle tissues. Scale bars: 50 μm. Quantitative analysis of microvessel density and the TUNEL assay (H and J). Area with yellow broken lines (C) indicates the infarct region. Red arrows (G and I) indicate microvessels and apoptotic cells, respectively. (K) Effective improvement of cardiac function by DKK2 injection. Cardiac functions were measured with 2D conventional parameters: FS and LVEF at 3 weeks after injection of DKK2 into MI rats. FS (%) = ([LVEDD – LVESD]/LVEDD) × 100 (%). LVEDV = 7.0 × LVEDD3/(2.4 + LVEDD), LVESV = 7.0 × LVESD3/(2.4 + LVESD), and LVEF (%) = (LVEDV – LVESV)/LVEDV × 100. *P < 0.05; **P < 0.01; ***P < 0.001. S; Sham, V; VEGF, D2; DKK2. Data represent mean ± SD.

(A–C) HUVECs stably expressing eGFP or DKK2 were transiently transfected with control, LRP5-specific, or LRP6-specific siRNA. After 60 hours, Cdc42 activity was measured (A). Cells were plated on Matrigel-coated plates at a density of 1.5 × 10⁵ cells/well and incubated for 18 hours. Microphotographs were taken and capillary-like networks were quantified with Image-Pro Plus software (B and C). (D) Coimmunoprecipitation of APC with Asef2. Lysates were prepared from cells overexpressing eGFP or DKK2, immunoprecipitated with anti-APC antibody, and probed with anti-Asef2 or anti-APC antibodies. (E) eGFP- or DKK2-expressing cells were transiently transfected with control or LRP6-specific siRNA. After 60 hours, cell lysates were subjected to coimmunoprecipitation. (F–H) eGFP- or DKK2-expressing cells were transiently transfected with control, APC-specific, or Asef2-specific siRNA. After 60 hours, Cdc42 activity was measured (F). The cells were plated on Matrigel-coated plates at a density of 1.5 × 10⁵ cells/well and incubated for 18 hours. Capillary-like networks were quantified with Image-Pro Plus software (G and H). Data represent mean ± SD. ***P < 0.001.