Angiogenin and Its Functions in Angiogenesis

The review is devoted to angiogenin, one of the factors that induce formation of blood vessels, which is unique in that it is a ribonuclease. Consideration is given to the tertiary structure of human angiogenin; the catalytic and cell receptor binding sites, their significance for angiogenic activity; the human angiogenin gene structure, chromosomal localization, and expression; the specificity of angiogenin as a ribonuclease and abolishment of protein synthesis; the nuclear localization of angiogenin in proliferating endothelial cells and its significance for angiogenic activity; angiogenin binding to cell surface actin as a plausible mechanism of inducing neovascularization (enhancement of plasminogen activation by actin, stimulation of the cell-associated proteolytic activity; promotion of the cultured cell invasiveness); modulation of mitogenic stimuli in endothelial, smooth muscle, and fibroblast cells by angiogenin. The importance of angiogenin as an adhesive molecule for endothelial and tumor cells is discussed too, as well as the modulation of tubular morphogenesis by bovine angiogenin, prevention of tumor growth in vivoby angiogenin antagonists, prospects of the use of angiogenin and angiogenin-encoding recombinant plasmids and vaccinia virus in therapeutic practice.     

Limited Proteolysis of Angiogenin by Elastase Is Regulated by Plasminogen

Human neutrophil elastase cleaves angiogenin at the Ile-29/Met-30 peptide bond to produce two major disulfide-linked fragments with apparent molecular weights of 10,000 and 4000, respectively. Elastase-cleaved angiogenin has slightly increased ribonucleolytic activity, but has lost its ability to undergo nuclear translocation in endothelial cells, a process essential for angiogenic activity. Cleavage appears to alter the cell-binding properties of angiogenin, despite the fact that it occurs some distance from the putative receptor-binding site, since the elastase-cleaved protein fails to compete with its native counterpart for nuclear translocation in endothelial cells. Plasminogen specifically accelerates elastase proteolysis of angiogenin. It does not enhance elastase activity toward ribonuclease A or the synthetic peptide substrate MeOSuc-Ala-Ala-Pro-Val-pNA. Plasminogen-accelerated inactivation of angiogenin by elastase might be a significant event in the process of angiogenin-induced angiogenesis since (i) angiogenin and plasminogen circulate in plasma at high concentrations, (ii) angiogenin, especially when bound to actin, activates tissue plasminogen activator to generate plasmin from plasminogen, and (iii) elastase cleaves plasminogen to produce angiostatin, a potent inhibitor of angiogenesis and metastasis. Interrelationships among angiogenin, plasminogen, plasminogen activators, elastase, and angiostatin may provide a sensitive regulatory system to balance angiogenesis and antiangiogenesis.     

Human angiogenin is rapidly translocated to the nucleus of human umbilical vein endothelial cells and binds to DNA

Human angiogenin is translocated to the nucleus of human umbilical vein endothelial cells in a time-dependent manner. Exogenous angiogenin appears in the nucleus in 2 min, reaches saturation in 15 min when 85% of the internalized angiogenin is in the nuclei, and remains associated with the nucleus for at least 4 h. Endothelial cells cultured at low density have a much higher capacity to translocate angiogenin to the nucleus than do those cultured at high density. This observation is consistent with previous findings that both the ability of endothelial cells to proliferate in response to angiogenin and the expression of an angiogenin receptor on the cell surface depend on cell density. Nuclear (125)I-angiogenin is not degraded and is neither spontaneously dissociated nor replaced by unlabeled angiogenin. It is, however, released by deoxyribonuclease I, but not by ribonuclease A, suggesting that angiogenin binds to DNA in the nucleus. These results suggest that in addition to acting as a ribonuclease, angiogenin may play a role in regulating gene expression by direct binding to DNA.     

Copper stimulates proliferation of human endothelial cells under culture

Copper ions stimulate proliferation of human umbilical artery and vein endothelial cells but not human dermal fibroblasts or arterial smooth muscle cells. Incubation of human umbilical vein endothelial cells for 48 h with 500 μM CuSO₄ in a serum-free medium in the absence of exogenous growth factors results in a twofold increase in cell number, similar to the cell number increase induced by 20 ng/ml of basic fibroblast growth factor under the same conditions. Copper-induced proliferation of endothelial cells is not inhibited by 10% fetal bovine serum or by the presence of antibodies against a variety of angiogenic, growth, and chemotactic factors including angiogenin, fibroblast growth factors, epidermal growth factor, platelet-derived growth factor, tumor necrosis factor-α, transforming growth factor-β, macrophage/monocyte chemotactic and activating factor, and macrophage inflammatory protein-1α. Moreover, despite the previous observations that copper increased total specific binding of ¹²⁵I-angiogenin to endothelial cells, binding to the 170 kDa receptor is not changed; hence, the mitogenic activity of angiogenin is not altered by copper. Copper-induced proliferation, along with early reports that copper induces migration of endothelial cells, may suggest a possible mechanism for the involvement of copper in the process of angiogenesis. J. Cell. Biochem. 69:326–335, 1998. © 1998 Wiley-Liss, Inc.     

Biological and structural features of murine angiogenin-4, an angiogenic protein

Murine angiogenin-4 (mAng-4) is a member of the pancreatic ribonuclease superfamily that is expressed in some endodermally derived organs. We now show that mAng-4 is angiogenic using a thoracic aorta assay never before applied to the angiogenins. mAng-4, human angiogenin (hAng), and murine angiogenin-1 (mAng-1) stimulate the proliferation of IGR1 melanoma cells but do not stimulate the proliferation or migration of bovine corneal endothelial cells or primary mouse embryonic fibroblasts. In addition, we report the 3-D structure of mAng-4 at 2.02-A resolution. The structure shows that the residues forming the putative B1, P1, and B2 RNA-binding subsites occupy positions similar to their hAng counterparts. The B1 subsite is obstructed by Glu115 and Ile118. The obstruction is stabilized by a novel salt bridge between the C-terminal carboxyl group and the side chain of Arg99. Through mutational studies, we identify residues critical to the angiogenic function of mAng-4. The effect of H12A and H112A mutations in the catalytic site indicates that ribonucleolytic activity is essential to angiogenesis. The consequences of a nearby E115A mutation are consistent with a significant role for Glu115 in the attenuation of enzymatic activity but also suggest that sufficient suppression of catalysis is necessary for angiogenesis. The effect of an R32A mutation in the putative nuclear localization sequence indicates that this residue is crucial for angiogenesis. In the putative cell-binding segment, the replacement of Lys59 with Asn (its counterpart at position 61 of hAng) does not abrogate enzymatic activity but abolishes angiogenic activity, the reason for which is unclear.     

Network of long-range concerted chemical shift displacements upon ligand binding to human angiogenin

Molecular recognition models of both induced fit and conformational selection rely on coupled networks of flexible residues and/or structural rearrangements to promote protein function. While the atomic details of these motional events still remain elusive, members of the pancreatic ribonuclease superfamily were previously shown to depend on subtle conformational heterogeneity for optimal catalytic function. Human angiogenin, a structural homologue of bovine pancreatic RNase A, induces blood vessel formation and relies on a weak yet functionally mandatory ribonucleolytic activity to promote neovascularization. Here, we use the NMR chemical shift projection analysis (CHESPA) to clarify the mechanism of ligand binding in human angiogenin, further providing information on long-range intramolecular residue networks potentially involved in the function of this enzyme. We identify two main clusters of residue networks displaying correlated linear chemical shift trajectories upon binding of substrate fragments to the purine- and pyrimidine-specific subsites of the catalytic cleft. A large correlated residue network clusters in the region corresponding to the V₁ domain, a site generally associated with the angiogenic response and structural stability of the enzyme. Another correlated network (residues 40–42) negatively affects the catalytic activity but also increases the angiogenic activity. ¹⁵N-CPMG relaxation dispersion experiments could not reveal the existence of millisecond timescale conformational exchange in this enzyme, a lack of flexibility supported by the very low-binding affinities and catalytic activity of angiogenin. Altogether, the current report potentially highlights the existence of long-range dynamic reorganization of the structure upon distinct subsite binding events in human angiogenin.     

Antiplasmin activity of a peptide that binds to the receptor-binding site of angiogenin.

It has been suggested that angiogenin binds to an actin-like molecule present on the surface of endothelial cells. Actin inhibits plasmin activity, but the angiogenin-actin complex is not active. In this report, we found that plasmin inhibits the interaction between angiogenin and actin suggesting a possibility that both angiogenin and plasmin may bind to a similar site on actin. Here we report that chANG, an antiangiogenin peptide that binds to the actin-binding site of angiogenin, inhibits the proteolytic activity of plasmin without any apparent effect on the activities of plasminogen activators and matrix metalloproteases. Its antiplasmin activity is comparable with that of actin. chANG inhibits plasmin activity via its binding to plasmin kringle domains while scrambled chANG does not bind to plasmin. chANG also inhibits the invasion of angiogenin-secreting human fibrosarcoma and colorectal carcinoma cells without effecting migration. Furthermore, chANG blocks angiogenesis induced by fibrosarcoma cells and metastasis of colorectal carcinoma cells to the liver. Therefore, the 11-amino acid peptide chANG has both antiangiogenin and antiplasmin activity, and could be useful in the development of anticancer agents.     

Replacement of residues 8-22 of angiogenin with 7-21 of RNase A selectively affects protein synthesis inhibition and angiogenesis.

The region of human angiogenin containing residues 8-21 is highly conserved in angiogenins from four mammalian species but differs substantially from the corresponding region of the homologous protein ribonuclease A (RNase A). Regional mutagenesis has been employed to replace this segment of angiogenin with the corresponding RNase A sequence, and the activities of the resulting covalent angiogenin/RNase hybrid, designated ARH-III, have been examined. The ribonucleolytic activity of ARH-III is unchanged toward most substrates, including tRNA, naked 18S and 28S rRNA, CpA, CpG, UpA, and UpG. In contrast, the capacity of ARH-III to inhibit cell-free protein synthesis is decreased 20-30-fold compared to that of angiogenin. The angiogenic activity of ARH-III is also different; it is actually more potent. It induces a maximal response in the chick chorioallantoic membrane assay at 0.1 ng per egg, a 10-fold lower dose than required for angiogenin. In addition, binding of ARH-III to the placental ribonuclease inhibitor is increased by at least 1 order of magnitude (Ki less than or equal to 7 x 10(-17) M) compared to angiogenin. Thus, mutation of a highly conserved region of angiogenin markedly affects those properties likely involved in its biological function(s); it does not, however, alter ribonucleolytic activity toward most substrates.     

Binding of human angiogenin inhibits actin polymerization

Angiogenin is a potent inducer of angiogenesis, a process of blood vessel formation. It interacts with endothelial and other cells and elicits a wide range of cellular responses including migration, proliferation, and tube formation. One important target of angiogenin is endothelial cell-surface actin and their interaction might be one of essential steps in angiogenin-induced neovascularization. Based on earlier indications that angiogenin promotes actin polymerization, we studied the binding interactions between angiogenin and actin in a wide range of conditions. We showed that at subphysiological KCl concentrations, angiogenin does not promote, but instead inhibits polymerization by sequestering G-actin. At low KCl concentrations angiogenin induces formation of unstructured aggregates, which, as shown by NMR, may be caused by angiogenin’s propensity to form oligomers. Binding of angiogenin to preformed F-actin does not cause depolymerization of actin filaments though it causes their stiffening. Binding of tropomyosin and angiogenin to F-actin is not competitive at concentrations sufficient for saturation of actin filaments. These observations suggest that angiogenin may cause changes in the cell cytoskeleton by inhibiting polymerization of G-actin and changing the physical properties of F-actin.     

Expression of receptors for human angiogenin in vascular smooth muscle cells.

Human angiogenin is a plasma protein with angiogenic and ribonucleolytic activities. Angiogenin inhibited both DNA replication and proliferation of aortic smooth muscle cells. Binding of 125I-angiogenin to bovine aortic smooth muscle cells at 4 degrees C was specific, saturable, reversible and involved two families of interactions. High-affinity binding sites with an apparent dissociation constant of 0.2 nm bound 1 x 104 molecules per cell grown at a density of 3 x 104.cm-2. Low-affinity binding sites with an apparent dissociation constant of 0.1 micrometer bound 4 x 106 molecules.cell-1. High-affinity binding sites decreased as cell density increased and were not detected at confluence. 125I-angiogenin bound specifically to cells routinely grown in serum-free conditions, indicating that the angiogenin-binding components were cell-derived. Affinity labelling of sparse bovine smooth muscle cells yielded seven major specific complexes of 45, 52, 70, 87, 98, 210 and 250-260 kDa. The same pattern was obtained with human cells. Potential modulators of angiogenesis such as protamine, heparin and the placental ribonuclease inhibitor competed for angiogenin binding to the cells. Together these data suggest that cultured bovine and human aortic smooth muscle cells express specific receptors for human angiogenin.     

Amino acid sequence of the nonsecretory ribonuclease of human urine

The amino acid sequence of a nonsecretory ribonuclease isolated from human urine was determined except for the identity of the residue at position 7. Sequence information indicates that the ribonucleases of human liver and spleen and an eosinophil-derived neurotoxin are identical or very closely related gene products. The sequence is identical at about 30% of the amino acid positions with those of all of the secreted mammalian ribonucleases for which information is available. Identical residues include active-site residues histidine-12, histidine-119, and lysine-41, other residues known to be important for substrate binding and catalytic activity, and all eight half-cystine residues common to these enzymes. Major differences include a deletion of six residues in the (so-called) S-peptide loop, insertions of two, and nine residues, respectively, in three other external loops of the molecule, and an addition of three residues at the amino terminus. The sequence shows the human nonsecretory ribonuclease to belong to the same ribonuclease superfamily as the mammalian secretory ribonucleases, turtle pancreatic ribonuclease, and human angiogenin. Sequence data suggest that a gene duplication occurred in an ancient vertebrate ancestor; one branch led to the nonsecretory ribonuclease, while the other branch led to a second duplication, with one line leading to the secretory ribonucleases (in mammals) and the second line leading to pancreatic ribonuclease in turtle and an angiogenic factor in mammals (human angiogenin). The nonsecretory ribonuclease has five short carbohydrate chains attached via asparagine residues at the surface of the molecule; these chains may have been shortened by exoglycosidase action.(ABSTRACT TRUNCATED AT 250 WORDS)     

Site-directed mutagenesis of histidine-13 and histidine-114 of human angiogenin. Alanine derivatives inhibit angiogenin-induced angiogenesis

The roles of His-13 and His-114 in the ribonucleolytic and angiogenic activities of human angiogenin have been investigated by site-directed mutagenesis. Replacement of either residue by alanine (H13A and H114A) decreases enzymatic activity toward tRNA by at least 10,000-fold and virtually abolishes 10,000-fold and virtually abolishes angiogenic activity in the chick embryo chorioallantoic membrane assay. Both the H13A and H114A mutant proteins compete effectively with angiogenin in the latter assay; only a 5-fold molar excess of H13A over unmodified protein is required for complete inhibition. The His----Ala substitutions, however, do not have any significant effect on the interaction of angiogenin with human placental ribonuclease inhibitor, an extremely potent inhibitor of angiogenin (Ki approximately 7 x 10(-16 M) previously shown to interact with another active-site residue, Lys-40. The effects of more conservative replacements-glutamine at position 13 and asparagine at position 114--were also examined. While the enzymatic activity of the H114N mutant was at least 3300-fold less than for the unmodified protein, the H13Q derivative had only 300-fold reduced activity toward tRNA and cytidylyl(3'----5') adenosine. Both substitutions substantially decreased angiogenic activity. The parallel effects on ribonucleolytic and biological activities observed with all four mutant proteins provide strong evidence that the latter activity of angiogenin is dependent on a functional enzymatic active site. The capacity of the H13A and H114A derivatives to compete with angiogenin in the chorioallantoic membrane assay suggests several additional features of the biological mode of action of this protein.     

Guest-host crosstalk in an angiogenin-RNase A chimeric protein

Angiogenin and ribonuclease A share 33% sequence identity but have distinct functions. Angiogenin is a potent inducer of angiogenesis that is only weakly ribonucleolytic, whereas ribonuclease A is a robust ribonuclease that is not angiogenic. A chimera ("ARH-I"), in which angiogenin residues 58-70 are replaced with residues 59-73 of ribonuclease A, has intermediate ribonucleolytic potency and no angiogenic activity. Here we report a crystal structure of ARH-I that reveals the molecular basis for these characteristics. The ribonuclease A-derived (guest) segment adopts a structure largely similar to that in ribonuclease A, and successfully converts this region from a cell-binding site to a purine-binding site. At the same time, its presence causes complex changes in the angiogenin-derived (host) portion that account for much of the increased ribonuclease activity of ARH-I. Guest-host interactions of this type probably occur more generally in protein chimeras, emphasizing the importance of direct structural information for understanding the functional behavior of such molecules.     

Base cleavage specificity of angiogenin with Saccharomyces cerevisiae and Escherichia coli 5S RNAs

The base cleavage specificity of angiogenin toward naturally occurring polyribonucleotides has been determined by using rapid RNA sequencing technology. With 5S RNAs from Saccharomyces cerevisiae and Escherichia coli, angiogenin cleaves phosphodiester bonds exclusively at cytidylic or uridylic residues, preferably when the pyrimidines are followed by adenine. However, not all of the existent pyrimidine bonds in the 5S RNAs are cleaved, likely owing to elements of structure in the substrate. Despite the high degree of sequence homology between angiogenin and ribonuclease A (RNase A), which includes all three catalytic as well as substrate binding residues, the cleavage patterns with natural RNAs are unique to each enzyme. Angiogenin significantly hydrolyzes certain bonds that are not appreciably attacked by RNase A and vice versa. The different cleavage specificities of angiogenin and RNase A may account for the fact that the former is angiogenic while the latter is not.     

Pro-angiogenic signaling by the endothelial presence of CEACAM1

Here, we demonstrate the expression of carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) in angiogenic sprouts but not in large mother blood vessels within tumor tissue. Correspondingly, only human microvascular endothelial cells involved in in vitro tube formation exhibit CEACAM1. CEACAM1-overexpressing versus CEACAM1-silenced human microvascular endothelial cells were used in migration and tube formation assays. CEACAM1-overexpressing microvascular endothelial cells showed prolonged survival and increased tube formation when they were stimulated with vascular endothelial growth factor (VEGF), whereas CEACAM1 silencing via small interfering RNA blocks these effects. Gene array and LightCycler analyses show an up-regulation of angiogenic factors such as VEGF, VEGF receptor 2, angiopoietin-1, angiopoietin-2, tie-2, angiogenin, and interleukin-8 but a down-regulation of collagen XVIII/endostatin and Tie-1 in CEACAM1-overexpressing microvascular endothelial cells. Western blot analyses confirm these results for VEGF and endostatin at the protein level. These results suggest that constitutive expression of CEACAM1 in microvascular endothelial cells switches them to an angiogenic phenotype, whereas CEACAM1 silencing apparently abrogates the VEGF-induced morphogenetic effects during capillary formation. Thus, strategies targeting the endothelial up-regulation of CEACAM1 might be promising for antiangiogenic tumor therapy.     

Angiogenin-induced protein kinase B/Akt activation is necessary for angiogenesis but is independent of nuclear translocation of angiogenin in HUVE cells

Angiogenin, a potent angiogenic factor, binds to endothelial cells and is endocytosed and rapidly translocated to and concentrated in the nucleolus where it binds to DNA. In this study, we report that angiogenin induces transient phosphorylation of protein kinase B/Akt in cultured human umbilical vein endothelial (HUVE) cells. LY294002 inhibits the angiogenin-induced protein kinase B/Akt activation and also angiogenin-induced cell migration in vitro as well as angiogenesis in chick embryo chorioallantoic membrane in vivo without affecting nuclear translocation of angiogenin in HUVE cells. These results suggest that cross-talk between angiogenin and protein kinase B/Akt signaling pathways is essential for angiogenin-induced angiogenesis in vitro and in vivo, and that angiogenin-induced PKB/Akt activation is independent of nuclear translocation of angiogenin in HUVE cells.     

Mutagenesis of residues flanking Lys-40 enhances the enzymatic activity and reduces the angiogenic potency of angiogenin.

The primary structure of the blood vessel inducing protein angiogenin is 35% identical with that of pancreatic ribonuclease (RNase) and contains counterparts for the critical RNase active-site residues His-12, Lys-41, and His-119. Although angiogenin is a ribonucleolytic enzyme, its activity toward conventional substrates is lower than that of pancreatic RNase by several orders of magnitude. Comparison of the amino acid sequences of RNase and angiogenin reveals several striking differences in the region flanking the active-site lysine, including a deletion and a transposition of aspartic acid and proline residues. In order to examine how these sequence changes alter the functional properties of angiogenin, an angiogenin/RNase hybrid protein (ARH-II), in which residues 38-41 of angiogenin (Pro-Cys-Lys-Asp) have been replaced by the corresponding segment of bovine pancreatic RNase (Asp-Arg-Cys-Lys-Pro), was prepared by regional mutagenesis. Compared to angiogenin, ARH-II has markedly diminished angiogenic activity on the chick embryo chorioallantoic membrane but 5-75-fold greater enzymatic activity toward a variety of polynucleotide and dinucleotide substrates. In addition, the specificity of ARH-II toward dinucleotide substrates differs from that of angiogenin and is qualitatively similar to that of pancreatic RNase. Thus, non-active-site residues near Lys-40 in angiogenin appear to play a significant role in determining enzymatic specificity and reactivity as well as angiogenic potency. An additional angiogenin/RNase hybrid protein (ARH-IV), in which residues 59-71 of ARH-II have been replaced by the corresponding segment of pancreatic RNase, was also prepared.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystallographic studies on structural features that determine the enzymatic specificity and potency of human angiogenin: Thr44, Thr80, and residues 38-41

Human angiogenin (Ang) is a potent inducer of blood vessel formation and is a member of the pancreatic ribonuclease superfamily. Its enzymatic activity is unusually weak and biased toward cleavage after cytidine nucleotides. As part of an ongoing investigation into the structural basis of Ang's characteristic activity, we have determined the crystal structures of three Ang variants having novel activity. (i) The structure of T44D-Ang indicates that Asp44 can participate directly in pyrimidine binding and that the intrinsic hydrogen-bonding capability of this residue largely governs the pyrimidine specificity of this variant. Unexpectedly, the mutation also causes the most extensive disruption of the C-terminus seen in any Ang variant thus far. This allows the side chain of Arg101 to penetrate the B(1) site, raising the possibility that it participates in substrate binding as occurs in ribonuclease 4. (ii) The structure of T80A-Ang supports the view that Thr80 plays little role in maintaining the obstructive conformation of the C-terminus and that its participation in a hydrogen bond with Thr44 selectively weakens the interaction between Thr44 and N3 of cytosine. (iii) ARH-II is an angiogenin/RNase A chimera in which residues 38-41 of Ang are replaced with the corresponding residues (38-42) of RNase A. Its structure suggests that the guest segment influences catalysis by subtle means, possibly by reducing the pK(a) of the catalytic lysine. The loss of angiogenic activity is not attributable to disruption of known cell-binding or nuclear translocation sites but may be a consequence of the chimera's enhanced ribonucleolytic activity.     

Suppression of uPA and uPAR Attenuates Angiogenin Mediated Angiogenesis in Endothelial and Glioblastoma Cell Lines

Background

In our earlier reports, we showed that downregulation of uPA and uPAR inhibited glioma tumor angiogenesis in SNB19 cells, and intraperitoneal injection of a hairpin shRNA expressing plasmid targeting uPA and uPAR inhibited angiogenesis in nude mice. The exact mechanism by which inhibition of angiogenesis takes place is not clearly understood.

Methodology/Principal Findings

In the present study, we have attempted to investigate the mechanism by which uPA/uPAR downregulation by shRNA inhibits angiogenesis in endothelial and glioblastoma cell lines. uPA/uPAR downregulation by shRNA in U87 MG and U87 SPARC co-cultures with endothelial cells inhibited angiogenesis as assessed by in vitro angiogenesis assay and in vivo dorsal skin-fold chamber model in nude mice. Protein antibody array analysis of co-cultures of U87 and U87 SPARC cells with endothelial cells treated with pU2 (shRNA against uPA and uPAR) showed decreased angiogenin secretion and angiopoietin-1 as well as several other pro-angiogenic molecules. Therefore, we investigated the role of angiogenin and found that nuclear translocation, ribonucleolytic and 45S rRNA synthesis, which are all critical for angiogenic function of angiogenin, were significantly inhibited in endothelial cells transfected with uPA, uPAR and uPA/uPAR when compared with controls. Moreover, uPA and uPAR downregulation significantly inhibited the phosphorylation of Tie-2 receptor and also down regulated FKHR activation in the nucleus of endothelial cells via the GRB2/AKT/BAD pathway. Treatment of endothelial cells with ruPA increased angiogenin secretion and angiogenin expression as determined by ELISA and western blotting in a dose-dependent manner. The amino terminal fragment of uPA down regulated ruPA-induced angiogenin in endothelial cells, thereby suggesting that uPA plays a critical role in positively regulating angiogenin in glioblastoma cells.

Conclusions/Significance

Taken together, our results suggest that uPA/uPAR downregulation suppresses angiogenesis in endothelial cells induced by glioblastoma cell lines partially by downregulation of angiogenin and by inhibition of the angiopoietin-1/AKT/FKHR pathway.