Factor-induced Angiogenesis Research Articles

Selective Binding of Endostatin Peptide 4 to Recombinant VEGF Receptor 3 In Vitro.

We previously reported that neostatin, a proteolytic fragment of collagen XVIII that includes endostatin, inhibits basic fibroblast growth factor-induced corneal angiogenesis and lymphangiogenesis. In experiments to determine which fragments in neostatin are responsible for binding to VEGF receptors (VEGFRs), we previously showed that a 28- mer sequence at the C-terminal of endostatin, known as endostatin peptide 9, preferentially binds VEGFR3-Fc over VEGFR1-Fc and VEGFR2-Fc. In the present study, we show that a different endostatin fragment, endostatin peptide 4 (26 mers long), also selectively binds VEGFR3-Fc and not VEGFR1-Fc or VEGFR2-Fc. From surface plasmon resonance data, the KD and Chi² (RU²) values for endostatin peptide 4 binding to VEGFR3-Fc are 5.72 × 10⁻⁸ M and 0.354, respectively. In conclusion, endostatin peptides 4 and 9 may be responsible for endostatin binding to VEGFR3-Fc, and this improved understanding of endostatin peptide binding to VEGFR3-Fc may support the development of therapeutics targeting lymphangiogenic processes.

Nitric oxide modulates hepatocyte growth factor/scatter factor-induced angiogenesis

There is limited knowledge about potential therapeutic targets in Hepatocyte growth factor/scatter factor (HGF)-induced pathophysiological angiogenesis. Recent candidates have included phosphatidylinositol-3-kinase, which is an upstream activator for endothelial nitric oxide (NO) synthase (NOS III). The current study is the first to evaluate the possible involvement of NOS-NO cascade in HGF-induced angiogenesis. NOS III inhibitors blocked the HGF-induced functional neovascularization in vivo, as quantified using vessel counts, 133Xe-clearance, and immunohistology. This was reversed by L-arginine. Western blot analysis of HGF-treated cells also revealed a temporal increase in HGF-induced phosphorylation. In a deconstructional approach, HGF induced the proliferation and chemokinesis of human endothelial cells. These phenotypic effects were inhibited by NOS inhibitors, L-NAME and L-NIO, and the NO scavenger, carboxy PTIO, but unaltered by 1400W, a NOS II inhibitor. This inhibition was reversed by spermine NONOate, a NO donor, which independently exerted a biphasic effect on endothelial cell proliferation. The modulation of NO did not alter HGF-induced chemoinvasion of endothelial cells, while spermine-NONOate destabilized HGF-induced tubulogenesis, suggesting that a single assay is not sufficient for predicting the final phenotypic outcome on angiogenesis. The study is the first to demonstrate that the NOS III nitric oxide is a key signal cascade in HGF-induced angiogenesis, and represents a promising target for the clinical management of pathological conditions characterized by overt HGF signaling.

Targeting of mitogen-activated protein kinases and phosphatidylinositol 3 kinase inhibits hepatocyte growth factor/scatter factor-induced angiogenesis.

Hepatocyte growth factor/scatter factor (HGF/SF) can sufficiently and independently induce pathophysiological angiogenesis. However, the treatment strategies have mostly been unsuccessful. The present study is the first to evaluate the possible targeting of downstream signals for the inhibition of HGF/SF-induced angiogenesis. In a multichannel scratch assay with human endothelial cells (ECs), HGF/SF induced a strong and prolonged activation of MAPK and cell proliferation that was inhibited by PD98059 and LY294002/wortmannin, selective inhibitors of MAPK and PI3K signaling modules, respectively. Western blotting demonstrated a temporal relation between the activation of the two pathways. Chemical inhibition of the PI3K and MAPK signals inhibited HGF/SF-induced chemoinvasion of ECs in vitro and blocked the HGF/SF-induced neovascularization into a polymer scaffold in vivo, as quantified by vessel counts and the clearance of radioactive 133Xe. These data indicate that MEK and PI3K inhibitors represent a promising approach to the clinical management of pathological conditions characterized by overt HGF/SF-induced angiogenesis.

Endothelial Nitric Oxide Synthase (eNOS) is not Required for Basic Fibroblast Growth Factor (bFGF) Induced Angiogenesis in vivo

s 987 termined. An in vitro assay was also done using Fgcoated wells, THP-1 cells, and peptide. In vitro, R1.l decreased monocyte adhesion to Fg by 74% compared with R1.2 (1433 ? 419 cells/well vs 5603 2 1204 cells/well). Balloon injury induced a more than fivefold increase in THP-1 adhesion compared with sham injury. Balloon-injured, R1.l-treated arteries demonstrated significantly less monocyte adhesion (12,036 -C 4938 cells/cm2 vs 22,080 ? 6408 cells/cm2) compared with R1.2. These data support a critical role for FgIMac-1 interactions on injured vascular surfaces and may provide an avenue for early modification of postinjury inflammatory and thrombotic cascades. Sidhu P. Gangadharan, MD Janet Plescia, BS Mohanned Eslami, MD Dario C. Altieri, MD Michael S. Conte, MD Brigham & Women's Hospital Boston, Mass ENDOTHELIAL NITRIC OXIDE SYNTHASE (ENOS) IS NOT REQUIRED FOR BASIC FIBROBLAST GROWTH FACTOR (BFGF) INDUCED ANGIOGENESIS IN VrVO Nitric oxide has been implicated as a crucial signaling molecule and regulator in angiogenesis, a process that requires endothelial cell proliferation, migration, and differentiation. bFGF is a potent angiogenic factor that has been shown to induce the cellular responses associated with angiogenesis, including endothelial cell division and migration. In vitro, bFGF increases NO production by stimulation of eNOS expression. However, the absolute requirement of eNOS for bFGF-induced angiogenesis has not been established. We approach this question by carrying out experiments using eNOS-/gene deficient mice. We injected 0.5 mL of growth factor reduced Matrigel mixed with and without bFGF (40 nglmL) subcutaneously into the abdomen of wild-type and eNOS-1mice. Matrigel, an artificial basement membrane matrix, was the substrate on which endothelial cells from surrounding vessels migrate, proliferate, and form capillaries. At 10 days, the Matrigel plugs were recovered, fixed, and processed for HE P < .001). Shear rate increased early but normalized by 2 weeks and was less than baseline at 8 weeks. Conclusion: This model of modestly increased collateral flow demonstrates arterial remodeling with increased arterial diameter, luminal area, wall thickness, and nuclear number. These structural changes are first seen at 1 to 2 weeks and are complete at 8 weeks. Because the early increase in medial thickness is not associated with an increase in cell nuclei, early remodeling may involve changes in extracellular matrix. Late remodeling may involve smooth