Helena Larsson

Structural basis and potential role of heparin/ heparan sulfate binding to the angiogenesis inhibitor endostatin

Recombinant mouse endostatin produced by mammalian cells was shown to bind to heparin with a Kd of 0.3 μM, suggesting that this interaction may play a role in its anti‐angiogenic activity. Alanine mutagenesis demonstrated that a major site of four clustered arginines (positions 155, 158, 184 and 270) and a second site (R193,R194) are essential for binding. The same epitopes also participate in endostatin binding to heparan sulfate and sulfatides but not in its binding to the extracellular protein ligands fibulin‐1 and fibulin‐2. Analyses with various heparin fragments demonstrated a minimum size (12mer) for efficient binding to endostatin and a crucial role of 2‐O‐ and 6‐O‐sulfation. Furthermore, a substantial proportion (10–50%) of heparan sulfate chains obtained from various tissues showed a distinct binding to endostatin, indicating its potential to interact with extracellular and/or membrane‐bound proteoglycans. Angiogenesis induced by basic fibroblast growth factor‐2 (FGF‐2), but not by vascular endothelial growth factor (VEGF), in a chick chorioallantoic membrane assay could be inhibited by endostatin in a dose‐dependent manner. The mutational block of heparin binding decreased endostatin inhibition to low levels but elimination of zinc binding had no effect.

Overexpression of VEGF 121 in Immortalized Endothelial Cells Causes Conversion to Slowly Growing Angiosarcoma and High Level Expression of the VEGF Receptors VEGFR-1 and VEGFR-2 in Vivo

Vascular endothelial growth factor (VEGF or vascular permeability factor) is an important angiogenic factor that is up-regulated in numerous benign and malignant disorders, including angiosarcoma, hemangiomas, and solid tumors. To determine the functional role of VEGF in the development of endothelial tumors, we expressed primate VEGF 121 in an endothelial cell line, MS1, derived from primary murine cells by immortalization with a temperature-sensitive SV40 large T antigen. This cell line expresses the VEGFR-2 (Flk-1/Kdr) receptor for VEGF. Expression of VEGF 121 led to the development of slowly growing endothelial tumors, which were histologically well-differentiated angiosarcomas. The angiosarcomas generated from MS1 VEGF cells demonstrated up-regulation of the VEGF receptors VEGFR-2 and VEGFR-1 (Flt-1) in vivo compared with benign hemangiomas generated from MS1 cells. Treatment of these cells with the VEGFR-2 tyrosine kinase inhibitor SU 1498 led to decreased expression of ets-1, a transcription factor which has been shown to be stimulated by VEGF. These results suggest that high level expression of VEGF in endothelial cells may result in malignant transformation. This transformation process likely involves both autocrine and paracrine pathways.

Fibroblast Growth Factor Receptor-1-mediated Endothelial Cell Proliferation Is Dependent on the Src Homology (SH) 2/SH3 Domain-containing Adaptor Protein Crk

Stimulation of fibroblast growth factor receptor-1 (FGFR-1) expressed on endothelial cells leads to cellular migration and proliferation. We have examined the role of the Src homology (SH) 2/SH3 domain-containing adaptor protein Crk in these processes. Transient tyrosine phosphorylation of Crk in fibroblast growth factor-2-stimulated endothelial cells was dependent on the juxtamembrane tyrosine residue 463 in FGFR-1, and a Crk SH2 domain precipitated FGFR-1 via phosphorylated Tyr-463, indicating direct complex formation between Crk and FGFR-1. Furthermore, Crk SH2 and SH3 domains formed ligand-independent complexes with Shc, C3G, and the Crk-associated substrate (Cas). Tyrosine phosphorylation of C3G and Cas increased as a consequence of growth factor treatment. We examined the role of Crk in FGFR-1-mediated cellular responses by use of cells expressing chimeric platelet-derived growth factor receptor-α/FGFR-1 (αR/FR) wild type and mutant Y463F receptors. The kinase activity of αR/FR Y463F was intact, but both Crk and the adaptor FRS-2 were no longer tyrosine-phosphorylated in the mutant cells. Both wild type and mutant receptor cells migrated efficiently, whereas cells expressing the mutant αR/FR Y463F failed to proliferate and Erk2 and Jun kinase activities were suppressed in these cells. In wild type αR/FR cells transiently expressing an SH2 domain mutant of Crk, Erk and Jun kinase activities as well as DNA synthesis were attenuated. Our data indicate that Crk participates in signaling complexes downstream of FGFR-1, which propagate mitogenic signals.

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.