Reza Forough

Overexpression of a secretory form of FGF-1 promotes MMP-1-mediated endothelial cell migration

A coordinated interaction between fibroblast growth factors (FGFs) and matrix metalloproteinases (MMPs) is implicated in migration of microvascular endothelial cells (ECs), an early stage of angiogenesis. Specifically, we investigated microvascular ECs migration in vitro, which can be initiated by the overexpression of a secretory form of the angiogenic fibroblast growth factor‐1 (FGF‐1) and mediated through the enzymatic activity of matrix metalloproteinase‐1 (MMP‐1). MMP‐1 is a member of the MMP family with a propensity for degradation of interstitial type I collagen. We stably overexpressed a chimeric FGF‐1 construct composed of the FGF‐4 signal‐peptide gene, linked in‐frame to the FGF‐1 coding frame gene (sp‐FGF‐1), in cultured postcapillary venular ECs. The presence of the biologically active form of FGF‐1 was readily detected in the conditioned medium of ECs transfected with sp‐FGF‐1 construct as demonstrated by DNA synthesis assay. The sp‐FGF‐1‐, but not the plasmid vector alone‐transfected ECs, exhibited an altered morphology as demonstrated by their conversion from a classic cobblestone form to a fibroblastlike shape that featured prominent neuritelike extensions. Addition of the anti‐FGF receptor 1 antibody (FGFR1 Ab) reverted the transformed phenotype of sp‐FGF‐1 transfectants. This suggests that the resulting phenotypic transformation in sp‐FGF‐1 transfectants requires an uninterrupted interaction between the FGF‐1 ligand and its receptor. We studied migration of cells through matrices of either highly pure collagen I or reconstituted basement membrane (matrigel) and found that sp‐FGF‐1‐transfected cells migrated two times and six times faster than the vector control transfectants in the respective matrices. We further demonstrated that the enhanced migration rate of sp‐FGF‐1‐transfected EC coincided with the induction of their MMP‐1 mRNA level and increased enzymatic activity. The enhanced migratory activity of sp‐FGF‐1 could be blocked with a selective inhibitor of MMP‐1. These results suggest that the multipotent FGF‐1 plays a key role in the early stages of angiogenesis, by mediating MMP‐1 proteolytic activity. J. Cell. Biochem. 78:487–499, 2000.

Long-term alcohol consumption increases matrix metalloproteinase-2 activity in rat aorta

Altered degradation of extracellular matrix (ECM) underlies vascular remodeling, a hallmark in the pathogenesis of cardiovascular diseases including hypertension and aneurysmal dilatation. Although alcohol is recognized as a risk factor for certain cardiovascular disease states, its role in vascular remodeling has not been completely explored. We studied the effect of chronic alcohol consumption on upregulation of the enzymatic activity of matrix metalloproteinase-2 (MMP-2) as a possible pathway for large vessel remodeling. For this purpose, female rats were placed on one of three diets: a modified Lieber-DeCarli liquid diet containing 35% ethanol-derived calories, a pair-fed liquid diet with ethanol replaced by isocaloric maltose-dextrin, or a standard rat pellet. Weekly blood alcohol concentration averaged 117+/-7.9 mg/dl for the alcohol-fed rats. At 2, 4, and 72 weeks, aortas were removed and processed for measuring MMPs activity by gelatin zymography. Aortic extracts from rats on long-term (72 weeks), but not the short-term (2 and 4 weeks), alcohol diets showed increased MMP-2 activity. Furthermore, histochemical analysis of the aortas showed distinct disruption of the elastic fibers only in the 72 weeks alcohol-fed rats, compared to the control animals. These observations demonstrate that long-term alcohol consumption up-regulates MMP-2 activity, which is coincident with the alteration of aortic ECM composition through the degradation of vascular elastin components.

Fibroblast Growth Factors, Fibroblast Growth Factor Receptors, Diseases, and Drugs

Maintenance of endothelial cells (ECs), the building blocks of the vascular tree, is a presumed function of fibroblast growth factors (FGFs). In particular, the two prototypic members of FGF family, namely FGF1 and FGF2, due to their potent mitogenic and pro-migratory activities, have the ability to induce metabolic and phenotypic changes in ECs that are required to stimulate angiogenesis. In addition to FGF1 and FGF2, 23 other members of the FGF family have since been identified and characterized and they are reviewed in relation to their disease pathology. Particular emphasis is given to the biology of the FGFs and FGFRs on how they mediate the onset of angiogenesis. The focus of the present review is to survey what is known about the role of the currently identified FGFs and their four high affinity tyrosine kinase receptors in diseases and the angiogenesis-targeted drugs currently in clinical trials. Some new and promising patented drugs that target the angiogenic pathway are discussed. Examination of the currently patented drugs may identify more potent and specific regulators of FGF/FGFR signaling system for treatment of tumor angiogenesis in clinical settings. Additionally, novel drug development strategies are highlighted and reviewed.

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.

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.

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. J. Cell. Biochem. 101: 1492–1504, 2007.