Journal: Gene therapy

Article Title: Recombinant AAV vector encoding human VEGF165 enhances wound healing.

doi: 10.1038/sj.gt.3301697

Figure Lengend Snippet: Figure 1 Induction of neo-angiogenesis by AAV-VEGF165. (a) Schematic representation of the two rAAV vectors used in this study and transducing the VEGF165 and the marker LacZ gene. TR, AAV terminal repeats; CMV, human cytomegalovirus immediate–early promoter; pA, polyadenylation site. (b) Expression of VEGF165 from AAV-VEFG165. Hamster CHO cells were mock treated or transduced with AAV-VEGF165 at a MOI of approxi- mately 100 particles/cell. After 24 h, cells were reacted with an anti-VEGF antibody by immunocytochemistry. (c) Chicken chorioallantoic membrane assay (CAM) angiogenic assay. CHO cells were mock transduced (left side) or transduced with AAV-VEGF and inoculated in a sponge implanted in the chorioallantoic membrane. Neo-angiogenesis was evaluated after 4 days from implantation.

Article Snippet: Samples were then rinsed in PBS and blocked with non-immune horse serum followed by incubation with a polyclonal rabbit anti-human VEGF antibody (1:500) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or mouse monoclonal anti- smooth muscle actin antibody (1:330) (Sigma, St Louis, MO, USA).

Techniques: Marker, Expressing, Transduction, Immunocytochemistry, Chick Chorioallantoic Membrane Assay, Membrane

Journal: Gene therapy

Article Title: Recombinant AAV vector encoding human VEGF165 enhances wound healing.

doi: 10.1038/sj.gt.3301697

Figure Lengend Snippet: Figure 2 Transduction of rat skin wounds with AAV-LacZ and AAV-VEGF165. (a) Expression of -galactosidase in rat skin wound. Recombinant AAV-LacZ (approximately 1011particles) was injected in rat skin wounds and -galactosidase activity was evaluated after 10 days. The arrow indicates intense blue color in the site of injection. (b) Histological examination of AAV-LacZ-injected skin wounds. High level expression of -galactosidase was detected in muscular cells of the panniculus carnosus up to 21 days after AAV-Lac-Z delivery. (c) Immunohistochemistry with anti-VEGF antibodies. Skin wounds were transduced with AAV-VEGF (approximately 1011 particles) and VEGF production was assessed by immunohistochemistry 21 days after treatment. High level expression of VEGF is detectable in muscular cells of panniculus carnosus.

Article Snippet: Samples were then rinsed in PBS and blocked with non-immune horse serum followed by incubation with a polyclonal rabbit anti-human VEGF antibody (1:500) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or mouse monoclonal anti- smooth muscle actin antibody (1:330) (Sigma, St Louis, MO, USA).

Techniques: Transduction, Expressing, Recombinant, Injection, Activity Assay, Immunohistochemistry

Journal: Gene therapy

Article Title: Recombinant AAV vector encoding human VEGF165 enhances wound healing.

doi: 10.1038/sj.gt.3301697

Figure Lengend Snippet: Figure 5 Histological examination of AAV-VEGF-165- and AAV-LacZ-transduced wounds. (a) Photomicrograph of AAV-LacZ-treated wound at day 9. The picture shows good epithelial and granulation tissue organization, with few capillaries in the superficial and deep dermis (hematoxylin/eosin; original magnification ×50). (b and c) Photomicrographs of AAV-LacZ-treated wound at day 18. A dense and well-structured granular tissue is evident, with few newly formed capillary vessels. Panel c shows detail of the granulation tissue (original magnification ×50). (d) Photomicrograph of AAV- VEGF165-treated wound at day 9. The picture shows intense epidermis and dermis remodeling, with numerous small vessels scattered throughout the wound area (hematoxylin/eosin; original magnification ×50). (e and f) Photomicrographs of AAV-VEGF165-treated wound at day 18. The picture shows complete remodeling of epithelium and dermis. Dense proliferation of newly formed capillaries, as well as vessels of greater diameter is observed, distributed in the entire wound area. Panel f shows a detail of the granulation tissue, displaying dense proliferation of fine and slightly congested small vessels (hematoxylin/eosin; original magnification ×50).

Article Snippet: Samples were then rinsed in PBS and blocked with non-immune horse serum followed by incubation with a polyclonal rabbit anti-human VEGF antibody (1:500) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or mouse monoclonal anti- smooth muscle actin antibody (1:330) (Sigma, St Louis, MO, USA).

Techniques:

Journal: Gene therapy

Article Title: Recombinant AAV vector encoding human VEGF165 enhances wound healing.

doi: 10.1038/sj.gt.3301697

Figure Lengend Snippet: Figure 6 6 Histological scores of wound healing after AAV-VEGF-165- or AAV-LacZ transduction. (a) Criteria to evaluate histological scores of wound healing. (b) Assessment of wound healing at 9 and 18 days after wounding and treatment with AAV-VEGF165 or AAV-LacZ, as indicated, according to the histological scores of panel a. #, statistical significance (P 0.05).

Article Snippet: Samples were then rinsed in PBS and blocked with non-immune horse serum followed by incubation with a polyclonal rabbit anti-human VEGF antibody (1:500) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or mouse monoclonal anti- smooth muscle actin antibody (1:330) (Sigma, St Louis, MO, USA).

Techniques: Transduction

Journal: Journal of the National Cancer Institute

Article Title: Effect of imatinib mesylate on neuroblastoma tumorigenesis and vascular endothelial growth factor expression.

doi: 10.1093/jnci/djh004

Figure Lengend Snippet: Fig. 4. Effect of imatinib on vascular endothelial growth factor (VEGF) expres- sion in neuroblastoma cells. A) SMS-KCNR cells were pre-incubated for 30 minutes with 1 or 15 M imatinib or medium (–), then incubated for 5 minutes in the presence ( ) or absence (–) of 50 ng/mL VEGF. Protein lysates were prepared, and 1 mg of each lysate was immunoprecipitated (IP) with an anti- Flk-1 antibody and immunoblotted (IB) with an anti-phospho-tyrosine antibody (pTy) or an anti-Flk-1 antibody. B) SMS-KCNR, SH-SY5Y, and NGP cells were plated on 100-mm dishes in medium containing 10% fetal calf serum and treated with 15 M imatinib for the indicated times. Total RNA was isolated, and 25 g of each RNA sample was subjected to northern blot analysis to detect VEGF mRNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. C) SMS-KCNR cells (2 106) were plated on 100-mm dishes and incubated for 24 hours with the indicated concentrations of imatinib. Protein lysates were pre- pared, and 40 g of each lysate was electrophoresed under nonreducing condi- tions and immunoblotted with an anti-VEGF antibody and an anti-actin anti- body. D) Neuroblastoma cells (2 105) were plated on 12-well plates, treated for 24 hours with 15 M imatinib (solid bars) or medium (shaded bars) in 10% serum-containing medium. The growth media were then collected from each well, and the concentration of VEGF in each medium was measured by an enzyme-linked immunosorbent assay. The number of viable cells in each well was determined by using the trypan blue dye exclusion assay.

Article Snippet: The concentration of VEGF protein was measured using enzyme-linked immunosorbent assay Quantikine kits (R&D Systems) with a mouse monoclonal antibody against human VEGF (R&D Systems), according to the manufacturer’s instructions.

Techniques: Incubation, Immunoprecipitation, Isolation, Northern Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay, Exclusion Assay

Journal: Journal of the National Cancer Institute

Article Title: Effect of imatinib mesylate on neuroblastoma tumorigenesis and vascular endothelial growth factor expression.

doi: 10.1093/jnci/djh004

Figure Lengend Snippet: Fig. 5. Effect of imatinib on in vivo neuroblastoma tumor growth. Intramuscular xenografts were established in SCID mice by injecting neuroblastoma SMS-KCNR cells. Seven days after the injection, mice received either imatinib at 100 mg/kg (n 10), imatinib at 200 mg/kg (n 10), or control vehicle (phosphate-buffered saline) alone (n 10) by oral gavage every 12 hours for 14 days. Tumors volumes were determined on day 14 of treatment, and the tumors were excised and used to make protein lysates. This experiment was repeated twice. Results of a representa- tive experiment are shown. A) Effect of imatinib at 100 mg/kg on the in vivo growth of neuroblastomas. B) A comparison of tumor volume among the three treatment groups. Representative data from two individual experiments are shown. The sym- bols represent the values determined for each animal receiving the indicated treat- ment, and the horizontal bars indicate the mean tumor volumes. *P.001 and **P .001; statistical significance was determined by one-way analysis of variance using a post-test Bonferroni comparison of groups (30). C) Upper panels: Protein lysates (40 g) obtained from four randomly selected xenograft tumors were electrophoresed and immunoblotted sequentially with an anti- VEGF antibody and an anti-actin antibody. Lower panel: Band densities were quantified by computed image analysis using NIH Image software (40). The relative levels of VEGF protein expressed in the tumors were normalized to the relative level of actin expression in the samples, and the mean value for the ratios of four representative tumor samples from the control group or the group treated with imatinib at 100 mg/kg is expressed in relative densitometric units (RDU). Error bars correspond to 95% confidence intervals. *P .028, Student’s unpaired t test.

Article Snippet: The concentration of VEGF protein was measured using enzyme-linked immunosorbent assay Quantikine kits (R&D Systems) with a mouse monoclonal antibody against human VEGF (R&D Systems), according to the manufacturer’s instructions.

Techniques: In Vivo, Injection, Control, Saline, Comparison, Software, Expressing