Basic Fibroblast Growth Factor–Induced Angiogenic Phenotype in Mouse Endothelium

Abstract

The mouse is the most commonly used species for in vivo studies on angiogenesis related to tumor development. Yet, to the best of our knowledge, very few reports on the in vitro interaction of the angiogenic basic fibroblast growth factor (bFGF) with mouse endothelial cells are available. Three mouse endothelial cell lines originated from aorta (MAECs), brain capillaries (MBECs), and heart capillaries (MHECs) were characterized for endothelial phenotypic markers, in vivo tumorigenic activity, and the capacity to respond in vitro to bFGF. These cells express angiotensin-converting enzyme, acetylated LDL receptor, constitutive endothelial nitric oxide synthase, and vascular cell adhesion molecule-1 and bind Griffonia simplicifolia-I lectin. When injected subcutaneously in nude mice, MAECs induced the appearance of slow-growing vascular lesions reminiscent of epithelioid hemangioendothelioma, whereas MBEC xenografts grew rapidly, showing Kaposi's sarcoma-like morphological features. No lesions were induced by injection of MHECs. MAECs, MBECs, and MHECs expressed both low-affinity heparan sulfate bFGF-binding sites and high-affinity tyrosine kinase receptors (FGFRs) on their surfaces. In particular, MAECs expressed FGFR-2/bek mRNA, whereas microvascular MBECs and MHECs expressed FGFR-1/flg mRNA. Accordingly, bFGF induced a mitogenic response and the phosphorylation of extracellular signal-regulated kinase-2 in all the cell lines. In contrast, upregulation of urokinase-type plasminogen activator expression was observed in bFGF-treated microvascular MBECs and MHECs but not in MAECs. Also, bFGF-treated MBECs and MHECs but not MAECs invaded a three-dimensional fibrin gel and formed hollow, capillary-like structures. The relevance of the modifications of the fibrinolytic balance of mouse microvascular endothelium in bFGF-induced angiogenesis was validated in vivo by a gelatin-sponge assay in which the plasmin inhibitors tranexamic acid and epsilon-aminocaproic acid given to mice in the drinking water inhibited neovascularization induced by the growth factor. In conclusion, differences in response to bFGF exist between large-vessel MAECs and microvascular MBECs and MHECs. Both in vitro and in vivo data point to a role of the profibrinolytic phenotype induced by bFGF in microvascular endothelial cells during mouse angiogenesis. Our observations make these endothelial cell lines suitable for further studies on mouse endothelium during angiogenesis and in angioproliferative diseases.

Gene expression profile in fibroblast growth factor 2-transformed endothelial cells

“…Moreover, FGF2-mediated GADDs induction occurs also at the protein level as con®rmed by Western blot analysis of the cell extracts and by immunostaining of newly formed blood vessels in s.c. FGF2-Matrigel plugs. In apparent contrast with these ®ndings, FGF2-T-MAE cells show a high proliferation rate (M Belleri, unpublished observation) and FGF2 induces DNA synthesis when administered to MAE cell cultures (Bastaki et al, 1997). Clearly, these observations point to a complex interplay among growth arresting genes and proto-oncogenes following FGF2 stimulation in endothelial cells.…”

“…Homology or identity for each clone was determined based on the highest score using Advanced Blast 2.0 (Altschul and Lipman, 1990) and reported in the third and fourth columns pZipFGF2-MAE cells cause the rapid growth of opportunistic vascular tumors when injected in nude mice (Gualandris et al, 1996). In contrast, parental MAE cells are poorly tumorigenic, giving raise to highly di erentiated, slow growing hemangiomas (Bastaki et al, 1997). To validate the involvement of candidate genes in endothelial cell transformation and angiogenesis, we performed semi-quantitative reverse transcribed-PCR analysis of pZipFGF2-MAE and MAE xenografts.…”

Fibroblast growth factor/fibroblast growth factor receptor system in angiogenesis

“…In vitro, endothelial cells of different origin express FGFR1 [9,10] and, under some circumstances, FGFR2 [11] whereas the expression of FGFR3 or FGFR4 has never been reported in endothelium.…”

Tumor microvasculature supports proliferation and expansion of glioma‐propagating cells

“…However, later passages of the tMVECs lost the surface expression of CD31, a phenomenon that has been noted before and was dependent on the migratory state of the cells and the density of the culture. 16,17 In another publication it has been suggested, that intracellular retention of the molecule could also account for a downregulation of surface-CD31. 13,18 Although the cells of the passages we used still showed the typical endothelial morphology, largely excluding overgrowth of the culture by contaminating cells, we determined the mRNA expression of a panel of markers.…”