FGFR1/PI3K/AKT signaling pathway is a novel target for antiangiogenic effects of the cancer drug fumagillin (TNP-470).

Fibroblast growth factor-1 (FGF1), a prototypic member of the FGF family, is a potent angiogenic factor. Although FGF-stimulated angiogenesis has been extensively studied, the molecular mechanisms regulating FGF1-induced angiogenesis are poorly understood in vivo. Fumagillin, an antiangiogenic fungal metabolite, has the ability to inhibit FGF-stimulated angiogenesis in the chicken chorioallantoic membrane (CAM). In the current study, chicken CAMs were transfected with a signal peptide-containing version of the FGF1 gene construct (sp-FGF1). Transfected CAMs were then analyzed in the presence and absence of fumagillin treatment with respect to the mRNA expression levels and protein activity of the FGF1 receptor protein (FGFR1), phosphatidylinositol 3-kinase (PI3K), and its immediate downstream target, AKT-1 (protein kinase B). Treatment of sp-FGF1-transfected CAMs with fumagillin showed downregulation for both PI3K and AKT-1 proteins in mRNA expression and protein activity. In contrast, no major alterations in FGFR1 mRNA expression level were observed. Similar patterns of mRNA expression for the above three proteins were observed when the CAMs were treated with recombinant FGF1 protein in place of sp-FGF1 gene transfection. Investigation using biotin-labeled fumagillin showed that only the FGF1 receptor protein containing the cytoplasmic domain demonstrated binding to fumagillin. Furthermore, we demonstrated endothelial-specificity of the proposed antiangiogenic signaling cascade using an in vitro system. Based on these findings, we conclude that the binding of fumagillin to the cytoplasmic domain of the FGF1 receptor inhibited FGF1-stimulated angiogenesis both in vitro and in vivo.

Urinary levels of matrix metalloproteinase 9 and 2 and tissue inhibitor of matrix metalloproteinase in patients with coronary artery disease.

OBJECTIVES: To evaluate the feasibility of an assay for urinary levels of matrix metalloproteinases (MMPs) and the potential usefulness of urinary MMPs as a marker of coronary atherosclerosis or acute coronary syndromes (ACS). METHODS AND RESULTS: We measured urine and plasma MMP-9, MMP-2 and urine tissue inhibitor of metalloproteinase (TIMP-1) in patients with ACS (n=27), patients with coronary artery disease (CAD), but no clinical instability (n=47) and a group of healthy volunteers (n=15) who were <35 years of age, had no risk factors for CAD and did not undergo angiography. Compared with volunteers, patients with ACS and CAD had higher urine MMP-9, urine TIMP-1, plasma MMP-9 and plasma MMP-2 levels, but these did not differ between those with CAD and ACS. Using the volunteers to roughly establish an upper limit of normal, 84% of the urine TIMP-1 values and 95% of the urine MMP-9 values were abnormally elevated among those with CAD and ACS. CONCLUSIONS: Urine MMP-9 and TIMP-1 levels are elevated in patients with CAD and ACS compared with healthy volunteers. A high percent of patients with CAD or ACS had elevated urine values of MMP-9 and TIMP-1 suggesting these variables might be a useful marker of atherosclerotic disease.

Transcription factor Ets-1 regulates fibroblast growth factor-1-mediated angiogenesis in vivo: role of Ets-1 in the regulation of the PI3K/AKT/MMP-1 pathway.

We previously demonstrated that a modified secreted form of fibroblast growth factor 1 (FGF-1), a prototypic member of the FGF family, has the ability to stimulate angiogenesis in an in vivo model of angiogenesis, the so-called chick chorioallantoic membrane assay or CAM. We recently defined the importance of the phosphatidylinositol 3-kinase/AKT pathway in FGF-1-mediated angiogenesis in this model using specific pharmacological inhibitors. In our continuing efforts to define the molecular signaling pathway regulating FGF-1-induced angiogenesis in vivo, we utilized a transcription factor activity assay and identified transcription factor Ets-1 as a critical effector of FGF-1-induced angiogenesis. Both activity and mRNA expression levels of the Ets-1 molecule were increased in response to FGF-1 overexpression in CAMs, as documented by electrophoretic mobility shift assay (gel shift) and reverse transcription real-time PCR techniques, respectively. Furthermore, the delivery of Ets-1 antisense (AS) into CAM tissues effectively reduced angiogenesis in the CAM assay. In addition, both Ets-1 AS-treated chicken CAMs and cultured endothelial cells exhibited a reduction in matrix metalloproteinase 1 gene expression levels. The Ets-1 AS-treated endothelial cells also demonstrated a reduction in migration. These data suggest that Ets-1 activation is a requisite for FGF-1-mediated angiogenesis in vivo. Therefore, Ets-1 might be a potential target for the generation of inhibitor drugs for the treatment of FGF-dependent pathological angiogenesis such as metastatic tumors, rheumatoid arthritis and diabetic retinopathy.

Phosphatidylinositide 3-kinase is important in late-stage fibroblast growth factor-1-mediated angiogenesis in vivo.

We previously reported that overexpression of a secreted version of fibroblast growth factor-1 (sp-FGF-1) has the ability to induce angiogenesis in the chicken chorioallantoic membrane (CAM). In our current study, we examine the effects of sp-FGF-1 through a time course analysis of angiogenesis in the chicken CAM on days 3, 4, and 5 after gene transfection. Significant angiogenesis was observed on days 4 and 5 after gene transfection in the CAM assay. To evaluate the role of phosphatidylinositide 3-kinase (PI3K) signaling in sp-FGF-1-induced angiogenesis, we analyzed mRNA expression levels of PI3K and protein activity through its immediate downstream target, AKT-1. We found upregulation of both PI3K and AKT mRNA expression levels in day 5 sp-FGF-1 versus day 5 vector control-transfected CAMs. Furthermore, by blocking PI3K phosphorylation using the specific inhibitor, LY294002, we found that downstream phosphorylation of AKT-1 was inhibited. More importantly, the blockade of the PI3K pathway via LY294002 in sp-FGF-1-transfected CAMs significantly inhibited angiogenesis. These results further elucidate the molecular mechanisms of the sp-FGF-1 signaling pathway and it underscores the importance of PI3K signaling in FGF-1-stimulated angiogenesis in vivo. It also provides a basis for the role of sp-FGF-1 in the development of therapeutic treatments to combat vascular insufficiencies and angiogenesis-dependent cancers.

Role of AKT/PKB signaling in fibroblast growth factor-1 (FGF-1)-induced angiogenesis in the chicken chorioallantoic membrane (CAM).

Transfection of chicken chorioallantoic membranes (CAMs) with a chimeric secreted version of fibroblast growth factor-1 (sp-FGF-1) gene construct leads to a significant increase in vascularization. Though FGF-stimulated angiogenesis has been extensively studied, the molecular mechanisms regulating FGF-1-induced angiogenesis are poorly understood in vivo. This study was designed to investigate the role of the AKT (PKB) kinase signaling pathway in mediating sp-FGF-1-induced angiogenesis in the chicken CAM. The involvement of the AKT pathway was demonstrated by up-regulation of AKT1 mRNA expression in sp-FGF-1 compared to vector alone control transfected CAMs as demonstrated by real-time RT-PCR. Western analysis using an antibody specific to the activated AKT (phosphorylated AKT), demonstrated an increase in AKT activity in sp-FGF-1 compared to vector control transfected CAMs. More importantly, the AKT inhibitor ML-9 significantly reduced sp-FGF-1-induced angiogenesis in CAMs. These results indicate that AKT signaling plays a role in FGF-1-stimulated angiogenesis in vivo and the AKT pathway may serve as a therapeutic target for angiogenesis-associated diseases.