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Mice overexpressing placenta growth factor exhibit increased vascularization and vessel permeability

¹ Laboratory of Molecular and Cell Biology, Istituto Dermopatico dell'Immacolata, IRCCS, 00167 Rome, Italy
² Transgenic Mice Service Center, Istituto Regina Elena, IRCCS, 00158 Rome, Italy

View larger version (134K): [in a new window]   Fig. 1. PlGF overexpression in the skin of transgenic mice. In situ hybridization analysis with an antisense PlGF riboprobe on the back skin of wild-type (A,B) and transgenic (C,D) mice. (A,C) Bright field; (B,D) dark field. Bars, 100 µm. (E) RNase protection assay for PlGF on total RNA from back skin biopsies of wild-type (WT) and transgenic (TG) littermates. The murine melanoma cell line M3 was used as a positive control for the endogenous PlGF transcript (E). (F) Western blot for PlGF on skin lysates. As a control, mouse recombinant PlGF (mrPlGF) was included. (G) ELISA for PlGF on 24-hour-conditioned medium of cultured keratinocytes from newborn transgenic and wild-type mice.

View larger version (129K): [in a new window]   Fig. 2. Hypervascularized skin phenotype of PlGF-transgenic mice. (A) Newborn mice are visibly redder than wild-type controls. (B) The redder color of the skin in transgenic mice persists in the adult (10-week-old). Enlarged vessels are evident in the ear of transgenic mice compared with controls. (C,D) Whole mount ear preparations from control (C) and transgenic (D) 10-week-old mice perfused with biotinylated-L. esculentum lectin showing the increase in vessel number, tortuosity and size in the transgenic ears. (E,F) Signs of active angiogenesis in transgenic mice: vessel spike emission (E, arrowheads), intussusception (E, arrows) and glomeruloid bodies (F). Bars, 100 µm.

View larger version (73K): [in a new window]   Fig. 3. Microlymphoangiography of the tail skin showing that in K14-PlGF-transgenic mice both lymphatic vessel diameters (vd) and mesh diameters (md) are comparable in size with those of wild-type mice.

View larger version (129K): [in a new window]   Fig. 4. Enhanced vascular permeability in K14-PlGF mice. (A,B) Evans blue dye-perfused transgenic mice show increased extravasated blue tracer in the skin compared with perfused control mice. (C,D) Whole mount ear preparations from 10-week-old mice perfused with biotinylated R. communis lectin. Numerous leakage sites are present in the ear vessels of transgenic mice (D, arrows). Bars, 100 µm. (E) Miles vascular permeability assay. 1-4: 25, 50, 125, 250 ng of injected PlGF, respectively; 5: site of PBS injection.

View larger version (140K): [in a new window]   Fig. 5. Ultrastructure of dermal capillaries in the skin of transgenic (B) and control (A) mice. The plasma membrane of endothelial cells (e) appears thinner and more irregular in transgenic mice compared with wild-type littermates. Several fenestrae (C, arrows) and gaps (D, arrowhead) are visible in the plasma membrane of transgenic mouse endothelial cells. p, pericyte. Bars, 1 µm.

View larger version (104K): [in a new window]   Fig. 6. Flt-1 and flk-1 induction in the skin of transgenic mice. In situ hybridization analysis for flt-1 and flk-1 in the skin of 15.5 d.p.c. embryos. The mRNA for flt-1 transmembrane receptor is undetectable in the skin of wild-type embryos (A,B) and is induced in the dermal endothelium of transgenic embryos (C,D). Flk-1 is transcribed in the skin endothelium of wildtype mice (E,F) and consistently upregulated in the dermal endothelium of transgenic embryos (G,H). (A,C,E,G) Bright field; (B,D,F,H) dark field. Bars, 40 µm. RNase protection assay for flt-1 (I) and flk-1 (J) confirmed that mRNA levels for both receptor are increased in the skin of transgenic 15.5 d.p.c. embryos compared with wild-type controls.