Takashi Kojima, Jin-Hong Chang, Dimitri T. Azar

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Presentation transcript:

Proangiogenic Role of ephrinB1/EphB1 in Basic Fibroblast Growth Factor-Induced Corneal Angiogenesis Takashi Kojima, Jin-Hong Chang, Dimitri T. Azar The American Journal of Pathology Volume 170, Issue 2, Pages 764-773 (February 2007) DOI: 10.2353/ajpath.2007.060487 Copyright © 2007 American Society for Investigative Pathology Terms and Conditions

Figure 1 EphrinB1 immunolocalization in normal and bFGF-induced neovascularized cornea. Uninjured normal eye was enucleated and stained with ephrinB1 (A) and propidium iodide (B). Overlay image was shown in C. After bFGF pellet (120 ng/pellet) insertion, mouse eyes were enucleated at days 1, 4, and 7 and stained with ephrinB1 (green) (D, G, and J) and CD31 (red) (E, H, and K) antibodies. Overlay images are shown in F, I, and L. Bar = 20 μm. Antibodies preincubated with cognate peptides were used as control (M–O). The American Journal of Pathology 2007 170, 764-773DOI: (10.2353/ajpath.2007.060487) Copyright © 2007 American Society for Investigative Pathology Terms and Conditions

Figure 2 The bFGF-induced vascularized cornea (day 7 after pellet insertion) was stained with ephrinB1 (A, D, and G) and vimentin (E), F4/80 (B), or Gr-1 (H). Overlay images are shown in C, F, and I. The ephrinB1 colocalized with vimentin (keratocyte marker) and Gr-1 (neutrophil marker) (F and I) but not with F4/80 (C). Bar = 20 μm. The American Journal of Pathology 2007 170, 764-773DOI: (10.2353/ajpath.2007.060487) Copyright © 2007 American Society for Investigative Pathology Terms and Conditions

Figure 3 EphB1 immunolocalization in normal and bFGF-induced neovascularized cornea. Uninjured normal eye was enucleated and stained with EphB1 (A) and propidium iodide (B). Overlay image was shown in C. After bFGF pellet (120 ng/pellet) insertion, mouse eyes were enucleated at days 1, 4, and 7 and stained with EphB1 (green) (D, G, and J) and CD31 (red) (E, H, and K) antibodies. Overlay images are shown in F, I, and L. Bar = 20 μm. Antibodies preincubated with cognate peptides were used as control (M–O). The American Journal of Pathology 2007 170, 764-773DOI: (10.2353/ajpath.2007.060487) Copyright © 2007 American Society for Investigative Pathology Terms and Conditions

Figure 4 Confocal micrographs of corneal neovascularization in wild-type mice 7 days after bFGF (120 ng/ml) pellet insertion. Immunohistochemical localization of EphB1 (green) (A and E), CD31 (red) (B and F), type IV collagen (blue) (C and G), and triple staining (D and H). Triple staining demonstrates EphB1-specific localization to the vascular endothelial region. I: Activation of pEphB1 on ephrinB1-Fc stimulation. EphB1 was stably expressed through the time course. The pEphB1 was detected faintly at 15 minutes after ephrinB1-Fc stimulation and then enhanced at 60 minutes. The American Journal of Pathology 2007 170, 764-773DOI: (10.2353/ajpath.2007.060487) Copyright © 2007 American Society for Investigative Pathology Terms and Conditions

Figure 5 Effect of ephrinB1-Fc on aortic ring assay. Aorta was embedded in type I collagen and then bathed in different media. Photographs were taken on days 1, 4, and 7 after embedding the aortas. All ephrinB1-Fc were clustered as described in Materials and Methods. Media: EBM only (A–C), 20 pmol (2 μg)/ml Fc (photograph is not shown), 20 pmol (2 μg)/ml ephrinB1-Fc (D–F), 50 ng/ml bFGF (G–I), 50 ng/ml bFGF with 20 pmol (2 μg)/ml Fc (photograph is not shown), and 50 ng/ml bFGF with 20 pmol (2 μg)/ml ephrinB1-Fc (J–L). M: Seven days after incubation, the number of microvessels was counted under the microscope. EphrinB1-Fc synergistically enhanced the tube formation. Results are representative of at least three independent experiments and represent the mean ± SEM. *P < 0.05. The American Journal of Pathology 2007 170, 764-773DOI: (10.2353/ajpath.2007.060487) Copyright © 2007 American Society for Investigative Pathology Terms and Conditions

Figure 6 Effect of ephrinB1-Fc on corneal pocket assay. The pellet containing ephrinB1-Fc (A–C), bFGF (50 ng/pellet) (D–F), bFGF + Fc (G–I), and ephrinB1-Fc + bFGF (J–L) was inserted into corneal stromal pocket as described in Material and Methods. Photographs were taken on days 1, 4, and 7 after implantation. bFGF-induced corneal neovascularization was significantly enhanced by ephrinB1-Fc in vivo. M: Seven days after pellet insertion, the area of corneal neovascularization was calculated using NIH ImageJ software. Results are representative of at least three independent experiments and represent the mean ± SEM. *P < 0.05. N: Activation of ERK by bFGF and ephrinB1-Fc. CPAE cell lysates were immunoblotted with anti-phospho-ERK1/2 antibody and anti-ERK1/2 antibody. When the cells were treated by the ephrinB1-Fc with bFGF, the activation of ERK was dramatically enhanced and sustained. The American Journal of Pathology 2007 170, 764-773DOI: (10.2353/ajpath.2007.060487) Copyright © 2007 American Society for Investigative Pathology Terms and Conditions