Cell-based and direct gene transfer-induced angiogenesis via a secreted chimeric fibroblast growth factor-1 (sp-FGF-1) in the chick chorioallantoic membrane (CAM) Reza Forough 1 , Xinyu Wang 1 , Luis A. Martinez-Lemus 1 , Dimitri Thomas 1 , Zhe Sun 1 , Kouros Motamed 2, *, Janet L. Parker 1 & Gerald A. Meininger 1 1 Department of Medical Physiology and Cardiovascular Research Institute, College of Medicine, The Texas A&M University System Health Science Center, College Station, Texas, USA; 2 Vascular Biology Center and Department of Pathology, Medical College of Georgia, Augusta, Georgia, USA Received 19 December 2002; accepted in revised form 21 May 2003 Key words: angiogenesis, CAM assay, FGF-1, gelatin sponge, signal peptide, transfection Abstract Fibroblast growth factor-1 (FGF-1) is a potent angiogenic factor; its structure lacks a signal peptide for secretion. We previously reported that the overexpression of a secreted version of FGF-1 (sp-FGF-1) in microvascular endothelial cells (ECs) enhances cell migration [Partridge et al. J Cell Biochem 2000; 78(3): 487]. In the current study, we have examined the angiogenic effects of sp-FGF-1 in chicken chorioallantoic membranes (CAMs). Two methods of examining the effects of sp-FGF-1 in CAMs were used: cell-mediated transfection via bovine ECs and direct gene transfection. In the cell-mediated gene transfection, those eggs that were implanted with a gelatin sponge seeded with ECs stably transfected to over-express sp-FGF-1 protein showed a significant increase in angiogenesis inside the sponge when compared to eggs treated with vector control-transfected ECs. In the direct gene transfer, eggs received sp-FGF-1 showed a significant increase in vascularization when compared to eggs received vector alone plasmids. These CAM models are useful both for studying molecular mechanisms of angiogenesis and for developing better gene therapy strategies. Introduction Angiogenesis is a morphological process by which new capillaries are formed from pre-existing blood vessels [1]. Physiological angiogenesis is brief and tightly regulated in the adult individual. In contrast, pathologic angiogenesis, such as that associated with progression of rheumatoid arthritis, diabetic retinopathy, and solid tumor metastatic cancers, is no longer self-limited. Our knowledge of the events leading to angiogenesis is greatly advanced by the existing in vivo experimental models such as chick CAM [2]. In addition, CAM assays have long served as popular in vivo bioassays for cataloging pro- and anti-angiogenic factors [3, 4]. In this regard, the angiogenic or anti-angiogenic ability of an added factor can be assessed through its stimulation or inhibition of angiogenesis in the host CAM. FGF-1 is one of the few polypeptide growth factors which has gained prominence as a potent stimulator of angiogenesis in the CAM assay [5, 6]. The angiogenic strength of FGF-1, a founding member of the FGF family, is demonstrated by the ability of this growth factor to influence each critical stage of angiogenesis, including EC detachment, migration, proliferation, and ultimate differ- entiation into a functional capillary vessel [7, 8]. The extent of FGF-1 influence on key steps of angiogenesis make this growth factor a potential candidate for therapeutic angiogenesis. Indeed, we previously reported enhanced migration of endothelial cells (ECs) transfected with a secreted version of FGF-1 (sp-FGF-1) chimeric gene construct [9]. This result supported our hypothesis that forced secretion of the FGF-1 protein, mediated by the ligated signal peptide to the N-terminus of the growth factor, increases delivery of the active growth factor from the transfected cells to the neighboring cells. In light of these in vitro results, we are currently developing in vivo models to evaluate the efficacy of the sp-FGF-1 as a potential drug for therapeutic angiogenesis. In the current study, we tested the angiogenic ability of the sp-FGF-1 construct using two delivery approaches: cell-based implant or naked DNA direct gene transfer in the CAM assay. For the first approach, we sought to develop an EC-based FGF-1 delivery approach because the strategic location of ECs at the interface of blood and tissue would permit efficient delivery of the therapeutic Correspondence to: Reza Forough, Department of Medical Physiology, College of Medicine, The Texas A&M University System Health Science Center, College Station, TX 77843, USA. Tel: þ1-979- 8457481; Fax: þ1-979-847-8635; E-mail: forough@tamu.edu Angiogenesis 6: 47–54, 2003. 47 Ó 2003 Kluwer Academic Publishers. Printed in the Netherlands.