Cell-Mediated Delivery of Fibroblast Growth Factor-2 and Vascular Endothelial Growth Factor onto the Chick Chorioallantoic Membrane: Endothelial Fenestration and Angiogenesis

Abstract

Fibroblast growth factor-2 (FGF2) and vascular endothelial growth factor (VEGF) exert their angiogenic activity by interacting with endothelial cells in a distinct manner. In this study, we investigated the morphological features of endothelial cells of the chick embryo chorioallantoic membrane (CAM) microvasculature after stimulation with FGF2 or VEGF. In order to provide a continuous delivery of the growth factor, we utilized a recently developed gelatin sponge/CAM assay in which a limited number of FGF2- or VEGF-transfected cells were adsorbed onto gelatin sponges and applied on the top of the CAM on day 8 of development. Their angiogenic activity was compared to that exerted by a single bolus of the corresponding growth factor. All the angiogenic stimuli induced a comparable vasoproliferative response, as demonstrated by the appearance of similar numbers of immature blood vessels within the sponge on day 12. No angiogenic response was observed in CAMs implanted with the corresponding parental cell lines or vehicle. Electron microscopy demonstrated that VEGF-overexpressing cells modified the phenotype of the endothelium of the blood vessels at the boundary between the implant and the surrounding CAM mesenchyme. The endothelial lining of 30% of these vessels showed segmental attenuations, was frequently interrupted and became fenestrated, mimicking what is observed in tumor vasculature. In contrast, the vessels consisted of continuous endothelium sealed by tight junctions in all the other experimental conditions. These results indicate that FGF2 and VEGF interact with endothelial cells of the CAM in a distinct manner. Both growth factors induce a potent angiogenic response, but only VEGF delivered in a continuous manner by its transfectants can modify the phenotype of the otherwise quiescent endothelium of CAM blood microvessels. The gelatin sponge/CAM assay may constitute a new model to study the mechanisms leading to endothelial fenestration in tumor growth.

Fibroblast growth factor/fibroblast growth factor receptor system in angiogenesis

“…Distinct patterns of vascular morphology upon FGF2 or VEGF stimulation are described also in the quail embryo CAM assay [86]. Finally, increased endothelial fenestration is observed in the blood vessels of the chick embryo CAM stimulated by VEGF-overexpressing cells, but not by FGF2-overexpressing cells, despite the quantitatively similar angiogenic response elicited by the two transfectants [84].…”

“…As compared to the application on the CAM of a single bolus of recombinant FGF2, cell implants overexpressing the growth factor allows the continuous delivery of FGF2 produced by a limited number of cells, thus mimicking more closely the initial stages of tumor angiogenesis [84]. Indeed, the release of 1.0 pg FGF2 per day from viable cells triggers an angiogenic response quantitatively similar to that elicited by 1.0 mg of the recombinant molecule [84]. Also, neutralizing anti-FGF2 antibodies prevent CAM neovascularization, supporting the key role of endogenous FGF2 in the development of vascular system in avian embryo [85].…”

45S5-Bioglass®-Based 3D-Scaffolds Seeded with Human Adipose Tissue-Derived Stem Cells Induce In Vivo Vascularization in the CAM Angiogenesis Assay

“…On the other hand a topic application of small amounts of positive regulators of angiogenesis directly onto scaffolds, for example, vascular endothelial growth factor (VEGF), has been shown to promote angiogenesis in the CAM assay. 26 However, this approach was unable to initiate a long-term angiogenic stimulus. Once the applied amount of growth factor was utilized, the angiogenic stimulus stopped.…”

Vascular Endothelial Growth Factor Localization in the Adult

“…10 Furthermore, VEGF has been shown to induce fenestrations in endothelial cells in vitro. [11][12][13] Fenestrated blood vessels are found in many secretory organs, including kidney, choroid plexus, and the choroid layer of the eye. Recent evidence has demonstrated a loss of tumor vessel fenestrations in pancreatic tumors after VEGF receptor blockade.…”