EGFL7 is a chemoattractant for endothelial cells and is up-regulated in angiogenesis and arterial injury

Abstract

The endothelium of the adult vasculature is normally quiescent, with the exception of the vasculature of the female reproductive system. However, in response to appropriate stimuli (ie, wound healing, atherosclerosis, tumor growth and metastasis, arthritis) the vasculature becomes activated and grows new capillaries through angiogenesis. We have recently identified a novel endothelial-restricted gene, Egfl7, that encodes a 41-kd secreted protein (Fitch MJ, Campagnolo L, Kuhnert F, Stuhlmann H: Egfl7, a novel epidermal growth factor-domain gene expressed in endothelial cells. Dev Dyn 2004, 230:316-324). Egfl7 is expressed at high levels early during mouse embryonic development and is strictly associated with the vascular bed. In this study, we investigated Egfl7 expression in the quiescent adult vasculature, in the pregnant uterus, and in two different models of arterial injury, namely ballooning and ferric chloride injury. By RNA in situ hybridization, Egfl7 expression in the vasculature was found to be restricted to the endothelium of the capillaries and mature vessels. In the pregnant uterus, increased vascularization was accompanied by up-regulation of Egfl7. On arterial injury, Egfl7 expression was up-regulated in the regenerating endothelium, but not in the neointima. Importantly, the EGFL7 protein acted as a chemoattractant for embryonic endothelial cells and fibroblasts in a cell migration assay. Together, these results suggest that Egfl7 functions in the formation and maintenance of endothelial integrity and that its up-regulation may be a critical component in the reorganization of the vascular bed in response to angiogenic stimuli.

Figure 1

Expression of Egfl7 in organ sections of adult CD-1 mice. A mouse-specific anti-sense Egfl7 [α-³⁵S]-UTP-labeled riboprobe, corresponding to the full-length Egfl7 cDNA, indicates levels of gene expression in the endothelium of liver (A, E), lung (B, F), outflow tract of the heart (C, G), and brain (D, H). Squares indicate regions magnified in E to H. Sections adjacent to A to D were immunostained for CD31/PECAM1 (I–L). All sections were counterstained with H&E, and photographed under dark field at ×20 (A–D) or bright field at ×63 magnification (E–L), respectively.

Figure 2

Expression of Egfl7 in pregnant and normal uterus. A–F: Normal and pregnant mouse uteri were paraffin-embedded, sectioned, and analyzed for EGFL7 expression both by RNA in situ hybridization and immunohistochemistry. A: Normal, nonpregnant uterus; B: 7.5-dpc pregnant uterus hybridized with the Egfl7 anti-sense riboprobe. D, E: CD31/PECAM1 staining of sections adjacent to A and B, respectively. C: Section adjacent to that shown in B, immunostained using an antibody against EGFL7. F: Section was incubated with generic rabbit IgG as a control for immunohistochemistry. Sections C to F were counterstained with H&E. G, H: Cryostat sections from normal, nonpregnant uterus were double immunostained for EphrinB2 (G) and EGFL7 (H). Images shown in G and H depict a peripheral area of the uterus. *, Uterus lumen. All sections were photographed using an Axiocam camera (Zeiss) under bright field (A–F) or dark field (G, H). Original magnifications: ×20 (A, B, D, E); ×40 (C, F, G, H).

Figure 3

Expression of Egfl7 and flk-1 in untreated control and balloon-injured rat arterial sections. A: Untreated control rat artery stained with H&E. B: Control sense Egfl7 riboprobe labeled with [³⁵S]-UTP on untreated control rat artery. C, E, F, G, and I: Mouse-specific anti-sense Egfl7 riboprobe labeled with [³⁵S]-UTP indicate levels of gene expression in an untreated control rat artery (C) and the balloon-injured rat arteries at 48 hours after injury (E), 2 weeks after injury (F, G), and 4 weeks after injury (I). D, H, J: Anti-sense flk-1 riboprobes labeled with [³⁵S]-UTP indicate gene expression in control rat arteries (D), 2 weeks after injury (H), and 4 weeks after injury (J). Photomicrographs under bright field (A) and under dark field (B–F, I, and J). G and H were stained with toluidine blue. Original magnifications: ×10 (A–F, I, J); ×40 (G, H).

Figure 4

Expression of Egfl7 in FeCl₃-denuded mouse carotid arteries. B, D, F–H, L, and N: Egfl7 RNA in situ hybridization on mouse carotids using the same probe as in Figures 1 and 2. P: A sense riboprobe was used as control. Sections adjacent to those used for in situ hybridization were immunostained for CD31/PECAM1 (A, C, E, G, I, M, Q, P). O: Generic rat IgG was used as control for immunohistochemistry. Q and P: Magnification of the areas delimited by the squares in C and E, respectively. Abbreviations on the bottom right-hand corner represent the number of days after the injury.

Figure 5

Expression of EGFL7 in human arterial plaques. Human-specific anti-sense EGFL7 riboprobe labeled with [³⁵S]-UTP indicates levels of gene expression in carotid artery samples from patients 7079 (A) and 2799 (B).

Figure 6

Cell migration studies. A: Detection of EGFL7 in culture supernatant and cell extracts from HEK293 cells transfected with a His-tagged Egfl7 vector, using immunoblotting with anti-His antibody. Two days after transfection, culture supernatant (lane 1) was harvested, and cell extract was prepared (lane 2). Media (900 μl) was TCA-precipitated, and the protein detected using an horseradish peroxidase-conjugated anti-(His)₆ antibody. B: MEFs (40,000 cells) were placed on the 8-μm (1 μg/ml FN-coated) trans-well inserts and inserted into wells that contained control medium, EGFL7-conditioned, or EGFL7-depleted media, respectively. Eighteen hours later, cells that had migrated to the lower side of the membrane were fixed, stained, and counted. Cell migration was determined from the average cell numbers in five fields per filter (counted at ×200 magnification) each from three independent experiments, each performed in duplicate and expressed as mean ± SD. Random cell migration, ie, migration in the absence of chemoattractant, was given the arbitrary value of 100%, and results are expressed as percent of control migration. C: Migration of C167 cells in response to EGFL7 is shown. Trans-well inserts (5 μm in diameter) were coated with 0.2% bovine serum albumin. D: Migration of RVSMC in response to EGFL7 is shown. Trans-well inserts (8 μm in diameter) were coated with 0.1 mg/dl FN. Methods and statistical evaluation in C and D were as described in B.