Generation of Biologically Active Angiostatin Kringle 1–3 by Activated Human Neutrophils

AbstractThe contribution of polymorphonuclear neutrophils (PMN) to host defense and natural immunity extends well beyond their traditional role as professional phagocytes. In this study, we demonstrate that upon stimulation with proinflammatory stimuli, human PMN release enzymatic activities that, in vitro, generate bioactive angiostatin fragments from purified plasminogen. We also provide evidence that these angiostatin-like fragments, comprising kringle domain 1 to kringle domain 3 (kringle 1–3) of plasminogen, are generated as a byproduct of the selective proteolytic activity of neutrophil-secreted elastase. Remarkably, affinity-purified angiostatin kringle 1–3 fragments generated by neutrophils inhibited basic fibroblast growth factor plus vascular endothelial growth factor-induced endothelial cell proliferation in vitro, and both vascular endothelial growth factor-induced angiogenesis in the matrigel plug assay and fibroblast growth factor-induced angiogenesis in the chick embryo chorioallantoic membrane assay, in vivo. These results represent the first demonstration that biologically active angiostatin-like fragments can be generated by inflammatory human neutrophils. Because angiostatin is a potent inhibitor of angiogenesis, tumor growth, and metastasis, the data suggest that activated PMN not only act as potent effectors of inflammation, but might also play a critical role in the inhibition of angiogenesis in inflammatory diseases and tumors, by generation of a potent anti-angiogenic molecule

The combination of Ezetimibe and Statin: a new treatment for hypercholesterolemia

The combination of Simvastatin and Ezetimibe allows dual inhibition of both cholesterol production and absorption. This treatment approach allows achieving same low serum cholesterol levels with the administration of much lower doses of statins. This should reduce side effects, compared to statin only therapy, enabling more patients to achieve their LDL cholesterol treatment goals. With ezetimibe/simvastatin therapy, reductions of about 60% from baseline in LDL cholesterol have been shown. Concomitant improvement in other lipid fractions have also been demonstrated. The ezetimibe/simvastatin combination has been well tolerated, with a safety profile similar to that of statin therapy. This article will review clinical experience with ezetimibe/simvastatin combination, commenting upon its place and potential value in the prevention of cardiovascular disease

Interaction of fibroblast growth factor-2 (FGF-2) with free gangliosides: Biochemical characterization and biological consequences in endothelial cell cultures. Molecular Biology of the Cell

Exogenous gangliosides affect the angiogenic activity of fibroblast growth factor-2 (FGF-2), but their mechanism of action has not been elucidated. Here, a possible direct interaction of sialo-glycolipids with FGF-2 has been investigated. Size exclusion chromatography demonstrates that native, but not heat-denatured, 125I-FGF-2 binds to micelles formed by gangliosides GT1b, GD1b, orGM1. Also, gangliosides protect native FGF-2 from trypsin digestion at micromolar concentrations, the order of relative potency being GT1b � GD1b � GM1 � GM2 � sulfatide � GM3 � galactosyl-ceramide, whereas asialo-GM1, neuraminic acid, and N-acetylneuramin-lactose were ineffective. Scatchard plot analysis of the binding data of fluorochrome-labeled GM1 to immobilized FGF-2 indicates that FGF–2/GM1 interaction occurs with a Kd equal to 6 �M. This interaction is inhibited by the sialic acid-binding peptide mastoparan and by the synthetic fragments FGF-2(112–129) and, to a lesser extent, FGF-2(130–155), whereas peptides FGF-2(10–33), FGF-2(39–59), FGF-2(86–96), and the basic peptide HIV-1 Tat(41–60) were ineffective. These data identify the COOH terminus of FGF-2 as a putative ganglioside-binding region. Exogenous gangliosides inhibit the binding of 125I-FGF-2 to high-affinity tyrosine-kinase FGF-receptors (FGFRs) of endothelial GM 7373 cells at micromolar concentrations. The order of relative potency was GT1b � GD1b � GM1 � sulfatide a � sialo-GM1. Accordingly, GT1b,GD1b, GM1, and GM2, but not GM3 an

Examining new models for the study of autocrine and paracrine mechanisms of angiogenesis through FGF2-transfected endothelial and tumour cells.

Angiogenesis is the process of generating new capillary blood vessels. Uncontrolled endothelial cell proliferation is observed in tumour neovascularization. Several growth factors and cytokines have been shown to stimulate endothelial cell proliferation in vitro and in vivo and among them FGF2 was one of the first to be characterised. FGF2 is a Mr 18,000 heparin-binding cationic polypeptide that induces proliferation, migration, and protease production in endothelial cells in culture and neovascularization in vivo. FGF2 interacts with endothelial cells through two distinct classes of receptors, the high affinity tyrosine-kinase receptors (FGFRs) and low affinity heparan sulfate proteoglycans (HSPGs) present on the cell surface and in the extracellular matrix. Besides experimental evidence for paracrine mode of action for FGF2, some observations raise the hypothesis that FGF2 may also play an autocrine role in endothelial cells. FGF2 may therefore represent a target for anti-angiogenic therapies. In order to assess the angiostatic potential of different classes of compounds, novel experimental models have been developed based on the autocrine and/or the paracrine capacity of FGF2

Substrate-immobilized HIV-1 Tat drives VEGFR2/αvβ3 integrin complex formation and polarization in endothelial cells

Objective—The HIV-1 transactivating factor (Tat) possesses features typical of both cell-adhesive and angiogenic growth factor (AGF) proteins, inducing endothelial cell (EC) adhesion and proangiogenic activation. Tat was exploited to investigate the events triggered by EC adhesion to substrate-bound AGF that lead to proangiogenic activation. Methods and Results—Immobilized Tat induces actin cytoskeleton organization, formation of avb3 integrin1focal adhesion plaques, and recruitment of vascular endothelial growth factor receptor-2 (VEGFR2) in the ventral plasma membrane of adherent ECs. Also, acceptor photobleaching fluorescence resonance energy transfer demonstrated that VEGFR2/avb3 coupling occurs at the basal aspect of Tat-adherent ECs. Cell membrane fractionation showed that a limited fraction of avb3 integrin and VEGFR2 does colocalize in lipid rafts at the basal aspect of Tat-adherent ECs. VEGFR2 undergoes phosphorylation and triggers pp60src/ERK1/2 activation. The use of lipid raft disrupting agents and second messenger inhibitors demonstrated that intact lipid rafts and the VEGFR2/pp60src/ERK1/2 pathway are both required for cytoskeleton organization and proangiogenic activation of Tat-adherent ECs. Conclusion—Substrate-immobilized Tat causes VEGFR2/avb3 complex formation and polarization at the basal aspect of adherent ECs, VEGFR2/pp60src/ERK1/2 phosphorylation, cytoskeleton organization, and proangiogenic activation. These results provide novel insights in the AGF/tyrosine kinase receptor/integrin cross-talk