Journal: Circulation Research

Article Title: Inhibition of Delta-Like-4–Mediated Signaling Impairs Reparative Angiogenesis After Ischemia

doi: 10.1161/circresaha.110.221663

Figure Lengend Snippet: Figure 1. Upregulation of Dll4 in ische- mic tissues. A, Time course of Dll4 tran- scription in skeletal muscles measured by quantitative PCR. *P0.05, ***P0.001 vs control (n6 each time point). B, Confocal images of adductor muscles showing the induction of Dll4 expression in newly forming capillaries, 5 days postischemia. Dll4 (green), IsolectinB4 (marker of ECs, red), DAPI (nucleus marker, blue). Scale bar: 100 m. Bot- tom images, Higher magnification images showing the high expression of Dll4 in new capillary sprouts. Scale bar: 50 m. C, Confocal images showing costaining of Dll4 (green) and PECAM-1 (EC marker, red). Scale bar: 100 m. Bottom images, High magnification of upper panels. Scale bar: 50 m. D, Dll4 expression in the myocardium 5 days after myocardial infarction. Scale bar: 50 m. The dotted line delimits the infarct border zone. Arrows point ECs double positive for Dll4 and PECAM-1.

Article Snippet: Briefly, following incubation with blocking buffer, the samples were incubated with primary antibody: Goat polyclonal anti-mouse Dll4 (1:25, R&D systems), Rat monoclonal anti-mouse CD31 (1:25, BD Bioscience), Goat anti-mouse Podocalyxin (1:100, R&D systems), Rabbit anti-NG2 (1:20, Chemicon), Rat monoclonal anti-mouse CD45 (1:50, BD Bioscience) or Rat monoclonal anti-mouse CD11b (1:50, R&D system) overnight at 4°C.

Techniques: Muscles, Real-time Polymerase Chain Reaction, Control, Expressing, Marker

Journal: Circulation Research

Article Title: Inhibition of Delta-Like-4–Mediated Signaling Impairs Reparative Angiogenesis After Ischemia

doi: 10.1161/circresaha.110.221663

Figure Lengend Snippet: Figure 2. Inhibition of Dll4-mediated signaling by Ad-sDll4. A, Western blot- ting showing the soluble protein of Dll4 in supernatant of Ad-sDll4–infected HUVECs. B, Real-time PCR results showing the induction of Hes-1 mRNA levels in HUVECs seeded on immobilized Dll4 and the inhibition of Hes-1 upregu- lation by coculture with HUVECs trans- fected with Ad-sDll4. Values are fold changes relative to Hes-1/-actin ratio in control (BSA, noninfected); n3 repli- cates. *P0.05, **P0.01. C, Represen- tative photograph of exposed hindlimb muscles showing distribution of -galactosidase (-gal) 5 days after Ad--gal injection (arrow shows site of femoral artery ligation; *adductor mus- cle). Right, Longitudinal section of adductor muscle showing -gal staining counterstained with eosin. Scale bar: 200 m. D, Time course of transgenic Dll4 mRNA levels in whole homogenized adductor muscles following Ad-sDll4 injection. E, Immunoblot represents the presence of extracellular domain of Dll4 (sDll4) (57 KDa only in Ad-sDll4–injected muscles). F, Induction of Notch target genes in adductor muscles at 3 days postischemia and inhibition of Hey-1 and Nrarp-1 upregulation by Ad-sDll4; §P0.001 vs contralateral muscles; *P0.05 vs Ad-Null; n6 each group.

Article Snippet: Briefly, following incubation with blocking buffer, the samples were incubated with primary antibody: Goat polyclonal anti-mouse Dll4 (1:25, R&D systems), Rat monoclonal anti-mouse CD31 (1:25, BD Bioscience), Goat anti-mouse Podocalyxin (1:100, R&D systems), Rabbit anti-NG2 (1:20, Chemicon), Rat monoclonal anti-mouse CD45 (1:50, BD Bioscience) or Rat monoclonal anti-mouse CD11b (1:50, R&D system) overnight at 4°C.

Techniques: Inhibition, Western Blot, Infection, Real-time Polymerase Chain Reaction, Control, Muscles, Injection, Ligation, Staining, Transgenic Assay

Journal: Circulation Research

Article Title: Inhibition of Delta-Like-4–Mediated Signaling Impairs Reparative Angiogenesis After Ischemia

doi: 10.1161/circresaha.110.221663

Figure Lengend Snippet: Figure 4. Dll4 Inhibition impairs muscle regeneration. A, Cross-section showing the whole perimeter of adductor muscles harvested at 14 days postischemia and stained with hematoxy- lin/eosin. Lower panel: higher magnification of upper panel. B) Considerable loss of muscle fibers in Ad-sDll4–injected ischemic muscles. Myocytes (phalloidin, green), microvessels (Isolect- inB4, red). Scale bar: 100 m. Bottom images, Lipid deposit is increased in Ad-sDll4–injected ischemic muscles as revealed by oil red O staining (red). Scale bar: 200 m. C, Quantification of muscle fibers, lipid degeneration, and necrosis area. *P0.05; n6 per group.

Article Snippet: Briefly, following incubation with blocking buffer, the samples were incubated with primary antibody: Goat polyclonal anti-mouse Dll4 (1:25, R&D systems), Rat monoclonal anti-mouse CD31 (1:25, BD Bioscience), Goat anti-mouse Podocalyxin (1:100, R&D systems), Rabbit anti-NG2 (1:20, Chemicon), Rat monoclonal anti-mouse CD45 (1:50, BD Bioscience) or Rat monoclonal anti-mouse CD11b (1:50, R&D system) overnight at 4°C.

Techniques: Inhibition, Muscles, Staining, Injection

Journal: Circulation Research

Article Title: Inhibition of Delta-Like-4–Mediated Signaling Impairs Reparative Angiogenesis After Ischemia

doi: 10.1161/circresaha.110.221663

Figure Lengend Snippet: Figure 6. Dll4 inhibition enhances leukocyte infiltration. A, Confocal microscopy images and bar graph show increased number of infiltrating leukocytes in Ad-sDll4–injected muscles (CD45, green; IsolectinB4, red). Contralateral normoperfused muscles are shown for reference (control). Scale bar: 100 m. ***P0.0001; n10 per group. B, High magnifications showing the linear alignment of leuko- cytes along the vessel long-axis in Ad-null–injected ischemic muscles and the loss of structured patterning in Ad-sDll4–injected ische- mic muscles. Scale bar: 50 m. C, Identification of CD11b leukocytes in ischemic muscles (CD11b, green; IsolectinB4, red). D, Flow cytometric analysis of single cell suspensions from skeletal muscle digests shows the increased abundance of CD45 leukocytes in Ad-sDll4–injected muscles. *P0.05; n6 per group. E, Confocal images and bar graph showing induction of leukocyte infiltration in normoperfused Ad-sDll4–injected muscles (CD45, green; IsolectinB4, red). *P0.001 vs Ad.gal; n6 per group.

Article Snippet: Briefly, following incubation with blocking buffer, the samples were incubated with primary antibody: Goat polyclonal anti-mouse Dll4 (1:25, R&D systems), Rat monoclonal anti-mouse CD31 (1:25, BD Bioscience), Goat anti-mouse Podocalyxin (1:100, R&D systems), Rabbit anti-NG2 (1:20, Chemicon), Rat monoclonal anti-mouse CD45 (1:50, BD Bioscience) or Rat monoclonal anti-mouse CD11b (1:50, R&D system) overnight at 4°C.

Techniques: Inhibition, Confocal Microscopy, Injection, Muscles, Control

Journal: Circulation Research

Article Title: Inhibition of Delta-Like-4–Mediated Signaling Impairs Reparative Angiogenesis After Ischemia

doi: 10.1161/circresaha.110.221663

Figure Lengend Snippet: Figure 7. Dll4 inhibition increases the release of CXCL1/IL-8. A, Ad-sDll4 increases the circulating levels of CXCL1 in mice with limb ischemia. *P0.05, **P0.001 vs Ad-Null; #P0.05, **P0.01 vs nonischemic mice (control); n6 each group. B, Left, IL-8 concentrations in condi- tioned media of THP1 monocytes cultured on immobilized Dll4 in the presence of TNF- or vehicle. To block Notch, DAPT or vehicle (DMSO) was added. Right, Hes1 expression levels in the same experiment. *P0.001 vs BSA; #P0.001 vs vehicle; n4. n.d. indicates not detectable. C, Left, IL-8 concentrations in conditioned media of THP1 monocytes cocultured with trans- fected HUVECs. Right, Hes1 expression levels in the same experiment. *P0.001 vs BSA; #P0.01 vs Ad-Null; n4. D, Time- lapse video microscopy images illustrating the endothelial network formation by HUVECs on Matrigel in the presence of DiI- labeled THP1 cells (red). HUVECs were transfected with Ad-Null or Ad-sDll4 (100 plaque-forming units/cell).

Article Snippet: Briefly, following incubation with blocking buffer, the samples were incubated with primary antibody: Goat polyclonal anti-mouse Dll4 (1:25, R&D systems), Rat monoclonal anti-mouse CD31 (1:25, BD Bioscience), Goat anti-mouse Podocalyxin (1:100, R&D systems), Rabbit anti-NG2 (1:20, Chemicon), Rat monoclonal anti-mouse CD45 (1:50, BD Bioscience) or Rat monoclonal anti-mouse CD11b (1:50, R&D system) overnight at 4°C.

Techniques: Inhibition, Control, Cell Culture, Blocking Assay, Expressing, Microscopy, Labeling, Transfection