Johan Dixelius

VEGF-receptor signal transduction

The family of vascular endothelial growth factors (VEGFs) currently includes VEGF-A, -B, -C, -D, -E, and placenta growth factor (PlGF). Several of these factors, notably VEGF-A, exist as different isoforms, which appear to have unique biological functions. The VEGF family proteins bind in a distinct pattern to three structurally related receptor tyrosine kinases, denoted VEGF receptor-1, -2, and -3. Neuropilins, heparan-sulfated proteoglycans, cadherins, and integrin alphavbeta3 serve as coreceptors for certain but not all VEGF proteins. Moreover, the angiogenic response to VEGF varies between different organs and is dependent on the genetic background of the animal. Inactivation of the genes for VEGF-A and VEGF receptor-2 leads to embryonal death due to the lack of endothelial cells. Inactivation of the gene encoding VEGF receptor-1 leads to an increased number of endothelial cells, which obstruct the vessel lumen. Inactivation of VEGF receptor-3 leads to abnormally organized vessels and cardiac failure. Although VEGF receptor-3 normally is expressed only on lymphatic endothelial cells, it is up-regulated on vascular as well as nonvascular tumors and appears to be involved in the regulation of angiogenesis. A large body of data, such as those on gene inactivation, indicate that VEGF receptor-1 exerts a negative regulatory effect on VEGF receptor-2, at least during embryogenesis. Recent data imply a positive regulatory role for VEGF receptor-1 in pathological angiogenesis. The VEGF proteins are in general poor mitogens, but binding of VEGF-A to VEGF receptor-2 leads to survival, migration, and differentiation of endothelial cells and mediation of vascular permeability. This review outlines the current knowledge about the signal transduction properties of VEGF receptors, with focus on VEGF receptor-2.

Ligand-induced Vascular Endothelial Growth Factor Receptor-3 (VEGFR-3) Heterodimerization with VEGFR-2 in Primary Lymphatic Endothelial Cells Regulates Tyrosine Phosphorylation Sites

Vascular endothelial growth factors (VEGFs) regulate the development and growth of the blood and lymphatic vascular systems. Of the three VEGF receptors (VEGFR), VEGFR-1 and -2 are expressed on blood vessels; VEGFR-2 is found also on lymphatic vessels. VEGFR-3 is expressed mainly on lymphatic vessels but it is also up-regulated in tumor angiogenesis. Although VEGFR-3 is essential for proper lymphatic development, its signal transduction mechanisms are still incompletely understood. Trans-phosphorylation of activated, dimerized receptor tyrosine kinases is known to be critical for the regulation of kinase activity and for receptor interaction with signal transduction molecules. In this study, we have identified five tyrosyl phosphorylation sites in the VEGFR-3 carboxyl-terminal tail. These sites were used both in VEGFR-3 overexpressed in 293 cells and when the endogenous VEGFR-3 was activated in lymphatic endothelial cells. Interestingly, VEGF-C stimulation of lymphatic endothelial cells also induced the formation of VEGFR-3/VEGFR-2 heterodimers, in which VEGFR-3 was phosphorylated only at three of the five sites while the two most carboxyl-terminal tyrosine residues appeared not to be accessible for the VEGFR-2 kinase. Our data suggest that the carboxyl-terminal tail of VEGFR-3 provides important regulatory tyrosine phosphorylation sites with potential signal transduction capacity and that these sites are differentially used in ligand-induced homo- and heterodimeric receptor complexes.

VEGF receptor-2 Y951 signaling and a role for the adapter molecule TSAd in tumor angiogenesis

Vascular endothelial growth factor receptor‐2 (VEGFR‐2) activation by VEGF‐A is essential in vasculogenesis and angiogenesis. We have generated a pan‐phosphorylation site map of VEGFR‐2 and identified one major tyrosine phosphorylation site in the kinase insert (Y951), in addition to two major sites in the C‐terminal tail (Y1175 and Y1214). In developing vessels, phosphorylation of Y1175 and Y1214 was detected in all VEGFR‐2‐expressing endothelial cells, whereas phosphorylation of Y951 was identified in a subset of vessels. Phosphorylated Y951 bound the T‐cell‐specific adapter (TSAd), which was expressed in tumor vessels. Mutation of Y951 to F and introduction of phosphorylated Y951 peptide or TSAd siRNA into endothelial cells blocked VEGF‐A‐induced actin stress fibers and migration, but not mitogenesis. Tumor vascularization and growth was reduced in TSAd‐deficient mice, indicating a critical role of Y951‐TSAd signaling in pathological angiogenesis.

Angiostatin and endostatin inhibit endothelial cell migration in response to FGF and VEGF without interfering with specific intracellular signal transduction pathways.

The anti‐angiogenic agents angiostatin and endostatin have been shown to affect endothelial cell migration in a number of studies. We have examined the effect of these agents on intracellular signalling pathways known to regulate endothelial cell migration and proliferation/survival. Both agents inhibited fibroblast growth factor (FGF)‐, and vascular endothelial growth factor (VEGF)‐mediated migration of primary human microvascular endothelial cells and affected vascular formation in the embryoid body model. However, using phosphospecific antibodies we could not detect any effect of angiostatin or endostatin on phospholipase C‐γ (PLC‐γ), Akt/PKB, p44/42 mitogen‐activated protein kinase (MAPK), p38 MAPK and p21‐activated kinase (PAK) activity. Furthermore, using a glutathione S‐transferase (GST)‐PAK pull‐down assay, we could not detect any effect on Rac activity. We conclude that angiostatin and endostatin inhibit chemotaxis, without affecting intracellular signalling pathways known to regulate endothelial migration and proliferation/survival.

A fragment of histidine-rich glycoprotein is a potent inhibitor of tumor vascularization

In this study, we show that recombinant human histidine-rich glycoprotein (HRGP) has potent antiangiogenic properties as judged from effects on a syngeneic tumor model in C57/bl6 mice. Growth of fibrosarcoma, a very aggressive tumor, was reduced by >60% by HRGP treatment, and tumor angiogenesis was dramatically decreased. Treatment with HRGP led to increased apoptosis and reduced proliferation in the tumors. In contrast, HRGP did not affect apoptosis or DNA synthesis in endothelial cells or tumor cells in vitro. The mechanism of action of HRGP involves rearrangement of focal adhesions and decreased attachment of endothelial cells to vitronectin and, as a consequence, reduced endothelial cell migration. By using truncated versions of HRGP, we demonstrate that the isolated 150 amino acid-residue His/Pro-rich domain, which is also released by spontaneous proteolysis from purified HRGP, mediates the inhibitory effect on chemotaxis. Moreover, the His/Pro-rich domain must be released from HRGP to exert its effect. This study shows for the first time inhibitory effects of HRGP on tumor vascularization in vivo, thus providing proof of concept that HRGP is an angiogenesis inhibitor.

Minimal Active Domain and Mechanism of Action of the Angiogenesis Inhibitor Histidine-Rich Glycoprotein

Histidine-rich glycoprotein (HRGP) is an abundant heparin-binding plasma protein that efficiently arrests growth and vascularization of mouse tumor models. We have shown that the antiangiogenic effect of HRGP is dependent on its histidine/proline-rich domain, which needs to be released from the mother protein to exert its effects. Here we identify a 35-amino-acid peptide, HRGP330, derived from the histidine/proline-rich domain as endowed with antiangiogenic properties in vitro and in vivo. The mechanism of action of HRGP330 involves subversion of focal adhesion function by disruption of integrin-linked kinase (ILK) and focal adhesion kinase (FAK) functions, inhibition of vascular endothelial growth factor (VEGF)-induced tyrosine phosphorylation of the FAK substrate alpha-actinin, and, as a consequence, an arrest in endothelial cell motility. The disturbed focal adhesion function is reflected in the ability of HRGP as well as of HRGP330 to prevent endothelial cell adhesion to vitronectin in a manner involving alpha(v)beta3 integrin. In conclusion, HRGP330, which we define as the minimal antiangiogenic domain of HRGP, exerts its effects through signal transduction targeting focal adhesions, thereby interrupting VEGF-induced endothelial cell motility.

Endostatin action and intracellular signaling: β-catenin as a potential target?

Abstract Endostatin, the C-terminal part of collagen XVIII, has been shown to inhibit blood vessel formation in different pathological conditions characterized by increased angiogenesis, such as growing tumors. Subcutaneous injection of endostatin in tumor-bearing mice leads to decreased tumor growth, and even in some cases, cure of tumor disease. Endostatin has been tested in a clinical phase I study and shown not to be toxic. Whether the finding in mice that endostatin treatment does not result in development of resistance will hold true in humans is too early to tell. Endostatin binds to a specific motif in heparan sulfate, which may serve a co-receptor function. The structure of a potential primary receptor is not known. The mechanism of action of endostatin in inhibition of angiogenesis and thereby, inhibition of tumor growth, involves apoptosis of tumor cells. The most consistent effect of endostatin on endothelial cells in vitro is inhibition of endothelial cell migration, which may be due to disturbed cell–matrix interactions. An interesting candidate for transducing endostatins effect on apoptosis and cell migration is β-catenin, an intracellular protein which participates both in cell adhesion and in transcriptional regulation.

The tyrosine kinase FRK/RAK participates in cytokine-induced islet cell cytotoxicity

Hallmarks of the inflammatory process in Type I diabetes are macrophage activation, local release of beta-cell-toxic cytokines and infiltration of cytotoxic T lymphocytes. We have observed recently that mice overexpressing active FRK (fyn-related kinase)/RAK (previously named GTK/Bsk/IYK, where GTK stands for gut tyrosine kinase, Bsk for beta-cell Src-homology kinase and IYK for intestinal tyrosine kinase) in beta-cells exhibit increased susceptibility to beta-cell-toxic events, and therefore, we now attempt to find a more precise role for FRK/RAK in these processes. Phosphopeptide mapping of baculovirus-produced mouse FRK/RAK revealed an autophosphorylation pattern compatible with Tyr-394 being the main site. No evidence for in vitro phosphorylation of the C-terminal regulatory sites Tyr-497 and Tyr-504 was obtained, nor was there any indication of in vitro regulation of FRK/RAK kinase activity. Screening a panel of known tyrosine kinase inhibitors for their ability to inhibit FRK/RAK revealed several compounds that inhibited FRK/RAK, with a potency similar to that reported for their ability to inhibit other tyrosine kinases. Cytokine-induced islet toxicity was reduced in islets isolated from FRK/RAK knockout mice and this occurred without effects on the production of nitric oxide. Addition of the nitric oxide inhibitor nitroarginine to FRK/RAK knockout islets exposed to cytokines decreased cell death to a basal level. In normal islets, cytokine-induced cell death was inhibited by the addition of two FRK/RAK inhibitors, SU4984 and D-65495, or by transfection with short interfering RNA against FRK/RAK. It is concluded that FRK/RAK contributes to cytokine-induced beta-cell death, and inhibition of this kinase could provide means to suppress beta-cell destruction in Type I diabetes.

Binding affinities of insulin-like growth factor-I (IGF-I) fusion proteins to IGF binding protein 1 and IGF-I receptor are not correlated with mitogenic activity

In this report, comparisons between molecular affinities and cellular proliferation activities have been made for insulin‐like growth factor‐I (IGF‐I) and two IGF‐I fusion proteins in order to evaluate fusion proteins as tools for receptor binding studies. Binding affinities and growth promoting effects of the N‐terminal fusion Z‐IGF‐I and the C‐terminal fusion IGF‐I‐Z, and native recombinant human IGF‐I, were analyzed. Binding kinetic properties of the three IGF‐I variants were analyzed using BIAcore kinetic interaction analysis testing for binding to both human IGF binding protein 1 (IGFBP‐1) and a soluble form of the human IGF type I receptor extracellular domains (sIGF‐IR). The growth promoting effects on SaOS‐2 human osteosarcoma cells of the different fusion proteins were analyzed. A comparison of receptor binding affinities and growth promoting effects shows that the fusion protein receptor affinity does not correlate with proliferative potential. The IGF‐I‐Z fusion, with the lowest receptor affinity, shows similar proliferative potential to native IGF‐I. However, the Z‐IGF‐I fusion protein, with twice the receptor affinity of IGF‐I‐Z, displays only about 70% of the IGF‐I‐Z growth promoting activity. Both IGF‐I fusion proteins possess similar affinity to IGFBP‐1. These results indicate that determinants other than the receptor affinity could be involved in the regulation of IGF‐I proliferative action. This study demonstrates that ligand fusion proteins may be useful to study mechanisms of ligand induced receptor activation.