ABSTRACT
Angiogenesis is one of the key processes in the growth and development of tumors. Class-3 semaphorins (Sema3) are characterized as axon guidance factors involved in tumor angiogenesis by interacting with the vascular endothelial growth factor signaling pathway. Sema3 proteins convey their regulatory signals by binding to neuropilins and plexins receptors, which are located on the effector cell. These processes are regulated by furin endoproteinases that cleave RXRR motifs within the Sema, plexin-semaphorins-integrin, and C-terminal basic domains of Sema3 protein. Several studies have shown that the furin-mediated processing of the basic domain of Sema3F and Sema3A is critical for association with receptors. It is unclear, however, if this mechanism can also be applied to other Sema3 proteins, including the main subject of this study, Sema3C. To address this question, we generated a variant of the full-length human Sema3C carrying point mutation R745A at the basic domain at the hypothetical furin recognition site 742RNRR745, which would disable the processing of Sema3C at this specific location. The effects produced by this mutation were tested in an in vitro angiogenesis assay together with the wild-type Sema3C, Sema3A, and Sema3F proteins. Our results showed that the inhibitory effect of Sema3C on microcapillary formation by human umbilical vein endothelial cells could be abrogated upon mutation at the Sema3C basic domain within putative furin cleavage site 742RNRR745, indicating that this site was essential for the Sema3 biological activity.
Subject(s)
Humans , Point Mutation/genetics , Angiogenesis Inhibitors/genetics , Semaphorins/genetics , Furin/genetics , Neovascularization, Pathologic/genetics , Plasmids , Reference Values , Time Factors , Transfection , Cell Line , Reverse Transcriptase Polymerase Chain Reaction , Angiogenesis Inhibitors/analysis , Semaphorins/analysis , Furin/analysis , Human Umbilical Vein Endothelial CellsABSTRACT
Endostatin, a carboxyl-terminal fragment of collagen XVIII is known as an anti-angiogenic agent, that specifically inhibits the proliferation of endothelial cell and the growth of several primary tumor. We report here the purification and characterization of the recombinant murine endostatin (rmEndostatin) which was expressed in a prokaryotic expression system. This rmEndostatin has similar physiochemical properties of yeast-produced recombinant endostatin, and it also specifically inhibits the proliferation and migration of bovine capillary endothelial cells stimulated by basic fibroblast growth factor. The biological activity of rmEndostatin was also shown by its anti-angiogenic ability on the chorioallantoic membrane of chick embryo in vivo. In this article, we demonstrate the refolding and purification of rmEndostatin, expressed using E. coli system, to a biologically active and soluble form. In addition, these results confirm the activity of endostatin as a potent anti-angiogenic agent. Copyright 2000 Academic Press.
Subject(s)
Cattle , Chick Embryo , Mice , Angiogenesis Inhibitors/pharmacology , Angiogenesis Inhibitors/isolation & purification , Angiogenesis Inhibitors/genetics , Animals , Blotting, Western , Cell Movement/drug effects , Chorion/pathology , Chorion/drug effects , Circular Dichroism , Collagen/pharmacology , Collagen/isolation & purification , Collagen/genetics , Electrophoresis, Polyacrylamide Gel , Endothelium, Vascular/drug effects , Endothelium, Vascular/cytology , Escherichia coli/genetics , Fibroblast Growth Factor 2/pharmacology , Neovascularization, Physiologic/drug effects , Peptide Fragments/pharmacology , Peptide Fragments/isolation & purification , Peptide Fragments/genetics , Protein Folding , Recombinant Proteins/pharmacology , Recombinant Proteins/isolation & purification , Recombinant Proteins/genetics , Solubility , Yeasts/geneticsABSTRACT
Angiostatin is a potent angiogenesis inhibitor that is composed of the first four kringles of plasminogen fragment. Angiostatin with one less kringle molecule (kringle 1 to 3) was recently demonstrated to be an effective angiogenic inhibitor. To determine whether recombinant plasminogen kringle 1-3 (rPK1-3) can inhibit the corneal neovascularization induced by potent angiogenic factors; angiogenin, bFGF, or VEGF, hydron polymer discs each containing 2.0 microg of angiogenin, 500 ng of bFGF, or 500 ng of VEGF respectively were implanted into the corneal stroma of 138 rabbit eyes, and then discs each containing 10 microg, 12.5 microg, 20 microg or 30 microg of rPK1-3 were implanted randomly. Discs containing phosphate buffered saline were also implanted as a control. The angiogenesis score on number and length of newly formed vessels on the each of the rabbit's cornea were recorded daily by two observers (blinded). The treated corneas were also examined histologically. Recombinant PK1-3 treated corneas showed less neovascularization induced by all angiogenic factors (p < 0.05). and the extent of inhibition of neovascularization was proportional to the concentration of rPK1-3 (p < 0.05). Histologic examination showed leukocyte infiltration into the corneal stroma on the PBS treated eyes whereas rPK1-3 treated eyes showed only traces of leukocytes. These results of the effective rPK1-3 inhibition of corneal neovascularization induced by angiogenin, bFGF, or VEGF suggest that this angiostatin related fragment, rPK1-3, may be useful in the treatment of various neovascular diseases. Copyright 2000 Academic Press.
Subject(s)
Chick Embryo , Rabbits , Angiogenesis Inhibitors/pharmacology , Angiogenesis Inhibitors/genetics , Animals , Chorion/drug effects , Chorion/blood supply , Cornea/pathology , Cornea/drug effects , Cornea/blood supply , Endothelial Growth Factors/pharmacology , Fibroblast Growth Factor 2/pharmacology , Kringles/genetics , Lymphokines/pharmacology , Microscopy/methods , Neovascularization, Pathologic/drug therapy , Plasminogen/pharmacology , Plasminogen/genetics , Recombinant Proteins/pharmacology , Recombinant Proteins/genetics , Ribonuclease, Pancreatic/pharmacologyABSTRACT
Endothelial cell proliferation and differentiation into blood capillaries (i.e., angiogenesis) are essential for growth and development, wound healing, osetogenesis, etc. But abnormal angiogenesis during tumor progression could lead to serious consequences. Angiogenesis is a complex biochemical process, and is often difficult to study the molecular mechanism in vivo due to interference by multitude of factors. Here, I present a non-transformed capillary endothelial cell line as a model which has been extensively characterized morphologically and biochemically to study the fundamentals of the angiogenic process. Studies completed in our laboratory also evidenced that expression of Glc3Man9GlcNAc2-PP-Dol is intricately connected with the balance between the cellular proliferation and apoptosis during angiogenesis.