The bone morphogenic protein antagonist gremlin is expressed during embryonic development and under different pathologic conditions, including cancer. Gremlin is a proangiogenic protein belonging to the cystine-knot superfamily that includes transforming growth factor- proteins and the angiogenic vascu- IntroductionThe bone morphogenic protein (BMP) antagonist gremlin 1 induces angiogenesis in a BMP-independent manner by binding to as-yetunidentified endothelial cell (EC) membrane receptors and activating multiple tyrosine kinase-dependent intracellular signaling pathways in ECs. 2,3 Gremlin is produced by human tumors 4,5 and is expressed by fibroblast growth factor-2 (FGF2)-activated ECs and tumor endothelium. 2 Thus, gremlin may play paracrine/autocrine roles in tumor neovascularization. The identification of the EC receptors activated by gremlin has so far been unsuccessful.Vascular endothelial growth factor receptor-2 (VEGFR2) is the major proangiogenic tyrosine kinase receptor expressed by ECs and is activated by different members of the vascular endothelial growth factor (VEGF) family. 6 Both gremlin and VEGFs belong to the cystine-knot protein superfamily, 7 suggesting possible structural and/or functional similarities among these proangiogenic factors. On this basis, we investigated the capacity of gremlin to interact with and activate VEGFR2. The results demonstrate that gremlin binds and activates VEGFR2, leading to VEGFR2-dependent angiogenic responses in vitro and in vivo. Methods Ligand-receptor interaction assaysInteraction of VEGF-A and gremlin (R&D Systems) with the immobilized extracellular domain of VEGFR2 (sVEGFR2; Calbiochem) was analyzed by surface plasmon resonance (BIAcore Inc) and by competitive enzymelinked immunosorbent assay (ELISA). VEGFR2 interaction on the EC surface was characterized by cross-linking experiments, whereas VEGFR2 dimerization was assessed by fluorescence resonance energy transfer analysis. In vitro angiogenic assaysMotility and 3-dimensional gel invasion assays were performed on human, murine, and bovine ECs. 3 When indicated, ECs were stably transfected with a pcDNA3.1 expression vector harboring the mouse VEGFR2 complementary DNA.
Basic fibroblast growth factor (FGF-2) interacts with high-affinity tyrosine-kinase fibroblast growth factor receptors (FGFRs) and low-affinity heparan sulfate proteoglycans (HSPGs) in target cells. Both interactions are required for FGF-2-mediated biological responses. Here we report the FGF-2 antagonist activity of novel synthetic sulfonic acid polymers with distinct chemical structures and molecular masses (MMs). PAMPS [poly(2-acrylamido-2-methyl-1-propanesulfonic acid)], (MM approximately 7,000-10,000), PAS [poly(anetholesulfonic acid)], (MM approximately 9,000-11,000), PSS [poly(4-styrenesulfonic acid)], (MM = 70,000), and poly(vinylsulfonic acid) (MM = 2,000), inhibited FGF-2 binding to HSPGs and FGFRs in fetal bovine aortic endothelial GM 7373 cells. They also abrogated the formation of the HSPG/FGF-2/FGFR ternary complex, as evidenced by their capacity to prevent FGF-2-mediated cell-cell attachment of FGFR-1-overexpressing, HSPG-deficient Chinese hamster ovary cells to wild-type HSPG-bearing cells. Direct interaction of the polysulfonates with FGF-2 was demonstrated by their ability to protect the growth factor from proteolytic cleavage. Accordingly, molecular modeling, based on the crystal structure of the interaction of FGF-2 with a heparin hexamer, showed the feasibility of docking PAMPS into the heparin-binding domain of FGF-2. In agreement with their FGF-2-binding capacity, PSS, PAS, and PAMPS inhibited FGF-2-induced cell proliferation in GM 7373 cells and murine brain microvascular endothelial cells. The antiproliferative activity of these compounds was associated with the abrogation of FGF-2-induced tyrosine phosphorylation of FGFR-1. Moreover, the polysulfonates PSS and PAS inhibited FGF-2-induced activation of mitogen-activated protein kinase-1/2, involved in FGF-2 signal transduction. In conclusion, sulfonic acid polymers bind FGF-2 by mimicking heparin interaction. These compounds may provide a tool to inhibit FGF-2-induced endothelial cell proliferation in angiogenesis and tumor growth.
Angiogenesis is the process of generating new capillary blood vessels. Uncontrolled endothelial cell proliferation is observed in tumor neovascularization and in angioproliferative diseases. Tumors cannot growth as a mass above few mm(3) unless a new blood supply is induced. It derives that the control of the neovascularization process may affect tumor growth and may represent a novel approach to tumor therapy. Angiogenesis is controlled by a balance between proangiogenic and antiangiogenic factors. The angiogenic switch represents the net result of the activity of angiogenic stimulators and inhibitors, suggesting that counteracting even a single major angiogenic factor could shift the balance towards inhibition. Heparan sulfate proteoglycans are involved in the modulation of the neovascularization that takes place in different physiological and pathological conditions. This modulation occurs through the interaction with angiogenic growth factors or with negative regulators of angiogenesis. Thus, the study of the biochemical bases of this interaction may help to design glycosaminoglycan analogs endowed with angiostatic properties. The purpose of this review is to provide an overview of the structure/function of heparan sulfate proteoglycans in endothelial cells and to summarize the angiostatic properties of synthetic heparin-like compounds, chemically modified heparins, and biotechnological heparins.
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