A Fragment of Histidine-Rich Glycoprotein Is a Potent Inhibitor of Tumor Vascularization

Abstract

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.

Activated Platelets Provide a Functional Microenvironment for the Antiangiogenic Fragment of Histidine-Rich Glycoprotein

“…Similar to PF4, the His/Pro-rich domain of HRG is capable of binding to heparan sulfate on the endothelial cell surface (27) and to interfere with VEGF-induced signaling and angiogenesis (24). However, the inhibition exerted by HRG is not due to a direct competition between HRG and the growth factor for access to heparan sulfate (18,24), as in the case of PF4. Instead, HRG targets focal adhesion function in endothelial cells and thereby prevents an angiogenic response to VEGF (38).…”

“…In our previous studies, we identified a proteolytically released 30-kDa fragment, derived from the His/Prorich domain of HRG, as mediator of its antiangiogenic effect (18). The fragment must be released from the full-length protein to be active and can be found in purified fractions of HRG in vitro.…”

“…We have previously shown that inhibition of VEGF-induced chemotaxis of TIME cells by HRGP335, a 25-amino acid synthetic peptide derived from the same region as HRGP330, is dependent on the presence of zinc (18,27). This assay was repeated using HRGP330 and human umbilical vein endothelial cells.…”

“…Histidine-rich glycoprotein (HRG; alternatively, HRGP/ HPRG) has been identified as an angiogenesis inhibitor in vitro and in vivo by us and others (17,18). HRG is a 75-kDa singlechain heparin-binding plasma protein produced by the liver (19).…”

“…We have previously shown that one of these fragments, a 30-kDa peptide derived from the histidine-and proline-rich (His/Pro-rich) domain of HRG, mediates the antiangiogenic effect of HRG and that this peptide must be released from the full-length protein to exert its effect as an inhibitor of angiogenesis (18). Moreover, in a screen for the minimal active antiangiogenic domain of HRG, using synthetically produced peptides covering the His/Pro-rich domain, we identified a 35-amino acid peptide (HRGP330) that retained the antiangiogenic properties in vitro and in vivo (24).…”