Tracking angiogenesis induced by skin wounding and contact hypersensitivity using a Vegfr2-luciferase transgenic mouse by Ning Zhang, Zuxu Fang, Pamela R. Contag, Anthony F. Purchio, and David B. West Blood Volume 103(2):617-626 January 15, 2004 ©2004 by American Society of Hematology

In vitro Vegfr2-luc expression and regulation. In vitro Vegfr2-luc expression and regulation. The p Vegfr2-luc transgene construct was transiently transfected into cells in culture, and transfection efficiency was normalized by cotransfection with pRL-TK renilla luciferase vector. (A) Comparison of luciferase expression in BAEC, LL/2, MLTC-1, T241, and HepG2 cells. (B) Transiently transfected BAECs were treated with angiogenesis inhibitor mithramycin (Mith), 2-methoxyestradiol (ME), or fumagillin (FU), as described in “Materials and methods.” C indicates control (C) Effect of cell proliferation on luciferase expression in BAEC and HeLa cells. Results are expressed as a percentage of luciferase activity relative to appropriate control. RLU indicates relative light unit. These experiments were repeated twice. Data are presented as mean ± SE. Ning Zhang et al. Blood 2004;103:617-626 ©2004 by American Society of Hematology

Vegfr2-luc expression during postnatal development. Vegfr2-luc expression during postnatal development. (A) Female Vegfr2-luc mice (n = 3) at the ages of 2, 3, 4, 6, 8, 10, and 15 weeks were imaged as described in “Materials and methods.” Luciferase signal was collected from the dorsal and ventral sides of the mice. The color overlay on the image represents the photons per second emitted from the animal, ranging from 104 to 106 photons/sec, as indicated by the color scales. (B) Quantification of luciferase signal from the whole body with LivingImage software (Xenogen). Luciferase activity is expressed per centimeter squared of animal surface area to correct for differences in animal sizes at different ages. Data are presented as mean ± SE (n = 3). Ning Zhang et al. Blood 2004;103:617-626 ©2004 by American Society of Hematology

Vegfr2-luc expression in different tissues. Vegfr2-luc expression in different tissues. (A) Female Vegfr2-luc mice (n = 3) were killed at 8 weeks of age. Selected organs were excised, homogenized, and measured for luciferase activity. Data are presented as mean ± SE. (B) Northern blot analysis of VEGFR2 mRNA expression. Total RNA (4 μg per lane) from various organs of 8-week-old female Vegfr2-luc mice was extracted and analyzed. Lower panel displays an image of ethidium-bromide–stained gel to demonstrate even loading. Ning Zhang et al. Blood 2004;103:617-626 ©2004 by American Society of Hematology

Vegfr2-luc expression during wound healing. Vegfr2-luc expression during wound healing. Punch wounds 8 mm in diameter were generated on the backs of female Vegfr2-luc mice (n = 12). The mice were injected intraperitoneally with luciferin and imaged for 5 minutes at selected time points. (A) The day 0 image was taken immediately after wounding, and subsequent images were taken on days 1, 4, 7, 10, 14, 17, and 21 after wounding. (B) Quantification of luciferase expression (photons/s) from the wound area with LivingImage software (Xenogen). Data are shown as mean ± SE. Ning Zhang et al. Blood 2004;103:617-626 ©2004 by American Society of Hematology

Inhibition of Vegfr2-luc expression and wound healing by DEX Inhibition of Vegfr2-luc expression and wound healing by DEX. (A) Punch wounds 8 mm in diameter were generated on the backs of female Vegfr2-luc mice (n = 8). Inhibition of Vegfr2-luc expression and wound healing by DEX. (A) Punch wounds 8 mm in diameter were generated on the backs of female Vegfr2-luc mice (n = 8). The mice were intraperitoneally injected with luciferin and imaged for 5 minutes at various time points after wounding. The day 0 image was taken immediately after wounding. Mice were intraperitoneally injected with DEX (10 mg/kg) or saline daily beginning 1 day before the wounding. (B) Quantification of luciferase expression (photons/sec) from the wound area with LivingImage software (Xenogen). Data are shown as mean ± SE. (C) Wound area was measured and shown as a percentage of the initial wound area on day 0. Data presented are mean ± SE. Ning Zhang et al. Blood 2004;103:617-626 ©2004 by American Society of Hematology

Histologic assessment of wound healing and response to DEX Histologic assessment of wound healing and response to DEX. Paraformaldehyde-fixed and stained sections are shown from the skin of nonwounded mice and from mouse wounds isolated 7 days after wounding in female Vegfr2-luc mice treated with saline or DEX (n =... Histologic assessment of wound healing and response to DEX. Paraformaldehyde-fixed and stained sections are shown from the skin of nonwounded mice and from mouse wounds isolated 7 days after wounding in female Vegfr2-luc mice treated with saline or DEX (n = 4 per group). Sections were taken vertically through the granulated tissue extending from the surface into the adipose and skeletal muscle. In the sections shown, the surface of the skin or granulated tissue is at the top of the image, and in some images the deeper adipose tissue and skeletal muscle are readily visible. (A) Blood vessels (red arrows) were visualized by staining with an anti-CD31 antibody. The granulation tissue of saline-treated wounds showed blood vessels that were of relatively small caliber. In DEX-treated wounds, the blood vessels in the granulated tissue were of larger caliber. Dermal blood vessels in the normal nonwounded skin tissue were substantially less dense. Original magnification, × 200. (B) Infiltration of leukocytes was visualized by staining with anti-CD11b antibody. There were few scattered leukocytes (red arrows) in normal nonwounded skin. Blue arrow points to hair follicles in normal skin. Saline-treated wounds had significant leukocyte infiltration that appeared to consist predominantly of macrophages. DEX-treated wounds had markedly less macrophage infiltration. Original magnification, × 200. (C) The presence of the VEGFR2 receptor in the tissue was assessed by immunohistochemical staining with an anti-VEGFR2 antibody. Compared with normal nonwounded skin tissue, saline-treated wounds showed increased staining in endothelial cells and in stromal cells. Staining intensity was higher in cells closest to the lesioned epithelium of the wound, with less staining in deeper tissue. Red arrows indicate blood vessels. DEX-treated wounds had less VEGFR2 staining. Original magnification, × 100. (D) At a higher magnification, the specific cell types that positively stained for VEGFR2 were more apparent. Red arrows indicate blood vessel endothelial cells, green arrows indicate stromal cells, and blue arrows indicate skeletal muscle cells. Original magnification, × 400. Ning Zhang et al. Blood 2004;103:617-626 ©2004 by American Society of Hematology

Dexamethasone inhibits Vegfr2-luc expression and ear swelling produced by CHS. (A) Two groups (n = 8) of female Vegfr2-luc mice were sensitized with oxazolone on day –6. Dexamethasone inhibits Vegfr2-luc expression and ear swelling produced by CHS. (A) Two groups (n = 8) of female Vegfr2-luc mice were sensitized with oxazolone on day –6. On day 0 the right ears were challenged with oxazolone and the left ears were treated with vehicle. Mice were intraperitoneally injected with DEX (10 mg/kg) or saline daily beginning 1 day before the oxazolone challenge. Imaging analysis was performed on the sensitization day (day –6) and daily after oxazolone challenge (day 0). (B) Quantification of luciferase expression (photons/sec) in all the ears with LivingImage software (Xenogen). Data are presented as mean ± SE (n = 8). (C) Ear thickness was measured daily with a micrometer. Data represent mean ± SE (n = 8). Ning Zhang et al. Blood 2004;103:617-626 ©2004 by American Society of Hematology

Histologic assessment of ears during induction of CHS and response to DEX. Paraformaldehyde-fixed serial sections of mouse ear were prepared from female Vegfr2-luc mice 2 days after challenge. Histologic assessment of ears during induction of CHS and response to DEX. Paraformaldehyde-fixed serial sections of mouse ear were prepared from female Vegfr2-luc mice 2 days after challenge. (A) H&E staining (original magnification ×400). Normal mouse ear has blood vessels (red arrows) in normal dermis. Blue arrows point to lymphatic vessels. After treatment with oxazolone, the ear was thickened because of the infiltration of inflammatory cells and edema. Dilated lymphatics (blue arrows) are also present after oxazolone treatment. Dermal vessels (red arrows) contain red blood cells and white blood cells. Ear cartilage, denoted by the letter C within this panel, is surrounded by dermal inflammatory infiltrate. An intraepidermal abscess (green arrow) is present, and the surrounding epidermis is hyperplastic. Yellow arrows identify sebaceous glands. Sensitized mice exposed to oxazolone and treated with DEX have minimal dermal inflammatory cell infiltration. (B) Giemsa staining, (original magnification × 400). Normal control skin has dermal mast cells (brown arrows) in association with the connective tissue surrounding ear cartilage. The ear treated with oxazolone is markedly thickened with prominent enlarged mast cells (brown arrows), dilated lymphatics (blue arrows), and dermal blood vessels (red arrows) in a hypercellular dermis containing a mixed population of inflammatory cells. An intraepidermal abscess (green arrow) is present, and the adjacent epidermis is hyperplastic. The ear treated with oxazolone and DEX resembles normal ear. (C) Staining with an anti-VEGFR2 antibody (original magnification × 400). Normal control ear shows positive peroxidase staining (indicated by the brown tint) in cells in the epidermis (black arrow) and sebaceous gland (yellow arrow). Ears treated with oxazolone show increased VEGFR2 staining in endothelial cells of blood vessels in the dermis (red arrow) and epidermis (black arrow). The oxazolone-treated ears also show some staining for VEGFR2 protein in skeletal muscle cells (purple arrow). Some dermal inflammatory cells also show staining. Epidermal hyperplasia is clearly shown. Generally, increased staining for VEGFR2 is found in the ears after oxazolone treatment; however, this is not specific for the endothelial cells. Ears treated with oxazolone and DEX have VEGFR2 staining in the epidermis (black arrow), endothelial cells of blood vessels (red arrow), and skeletal muscle cells (purple arrows). Overall VEGFR2 staining is reduced in ear tissues of DEX-treated mice. Ning Zhang et al. Blood 2004;103:617-626 ©2004 by American Society of Hematology

Evaluation of intraepidermal and dermal inflammation. Evaluation of intraepidermal and dermal inflammation. (A) Epidermal accumulation of polymorphonuclear neutrophils (PMNs) and epidermal hyperplasia were quantified. Data are expressed as mean ± SE (n = 4). (B) Dermal PMNs, mast cells, and edema were quantified. Data are expressed as mean ± SE (n = 4). Ning Zhang et al. Blood 2004;103:617-626 ©2004 by American Society of Hematology