Peptides derived from the histidine-proline domain of the histidine-proline-rich glycoprotein bind to tropomyosin and have antiangiogenic and antitumor activities.

The antiangiogenic activity of the multidomain plasma protein histidine-proline-rich glycoprotein (HPRG) is localized to its histidine-proline-rich (H/P) domain and has recently been shown to be mediated, at least partially, through binding to cell-surface tropomyosin in fibroblast growth factor-2-activated endothelial cells (X. Guan et al., Thromb Haemost, in press). HPRG and its H/P domain, but not the other domains of HPRG, bind specifically and with high affinity to tropomyosin. In this study, we characterize the interaction of the H/P domain with tropomyosin and delineate the region within the H/P domain responsible for that interaction. The H/P domain of HPRG consists mostly of repetitions of the consensus sequence [H/P][H/P]PHG. Applying an in vitro tropomyosin binding assay, we demonstrate that the synthetic peptide HHPHG binds to tropomyosin in vitro and inhibits angiogenesis and tumor growth in vivo. The affinity for tropomyosin increases exponentially upon multimerization of the HHPHG sequence, with a concurrent increase in antiangiogenic activity. Specifically, the tetramer (HHPHG)4 has significant antiangiogenic activity in the Matrigel plug model (IC50 approximately 600 nm) and antitumor effects in two syngeneic mouse tumor models. Thus, we show that a 16-mer peptide analogue mimics the antiangiogenic activity of intact HPRG and is also able to inhibit tumor growth, suggesting that cell surface tropomyosin may represent a novel antiangiogenic target for the treatment of cancer.

Histidine-proline rich glycoprotein (HPRG) binds and transduces anti-angiogenic signals through cell surface tropomyosin on endothelial cells.

The anti-angiogenic properties of the histidine-proline-rich (H/P) domain of HPRG have recently been described (Juarez JC, et al. Cancer Research 2002; 62: 5344-50). However, the binding site that mediates these properties is unknown. HPRG is evolutionarily, functionally and structurally related to cleaved high molecular weight kininogen (HKa), an anti-angiogenic polypeptide that stimulates apoptosis of proliferating endothelial cells through binding to cell-surface tropomyosin (Zhang J-C, et al. Proc Natl Acad Sci USA 2002; 99: 12224-9). In this study, we demonstrate that HPRG binds with high affinity to FGF-2-stimulated human umbilical vein endothelial cells (HUVEC) and immobilized tropomyosin in a Zn2+ or pH-dependent manner, and that this interaction is mediated by the H/P domain of HPRG. At least two binding sites for HPRG, tropomyosin and heparan sulfate proteoglycans (HSPs), were identified on the surface of FGF-2-activated endothelial cells. Translocation of tropomyosin to the surface of HUVEC occurred in response to FGF-2, and the anti-angiogenic activity of HPRG in a Matrigel plug model was partially inhibited by soluble tropomyosin. These results suggest that HPRG binds to endothelial cell surface tropomyosin which at least partially mediates the antiangiogenic effects of HPRG.

Histidine-proline-rich glycoprotein has potent antiangiogenic activity mediated through the histidine-proline-rich domain.

Histidine-proline-rich glycoprotein (HPRG) is an abundant multidomain plasma protein evolutionarily related to high-molecular-weight kininogen. The cleaved form of high-molecular-weight kininogen has recently been demonstrated to exhibit antiangiogenic activities in vitro (J. C. Zhang et al., FASEB J., 14: 2589-2600, 2000), mediated primarily through domain 5. HPRG contains a histidine-proline-rich (H/P) domain with sequence and functional similarities to HKa-D5. We hypothesized that HPRG may also have antiangiogenic properties, localized within its H/P domain. The H/P domain is highly conserved among species, and because rabbit H/P domain is more resistant to internal proteolytic cleavage than the human domain, the rabbit HPRG (rbHPRG) was primarily used to assess the antiangiogenic activity of HPRG. Rabbit HPRG inhibited human umbilical vein endothelial cell (HUVEC) tube formation stimulated by fibroblast growth factor-2 (FGF-2) or vascular endothelial growth factor on a Matrigel surface as well as cell proliferation of FGF-2 stimulated HUVECs. The antiangiogenic activity of rbHPRG was localized to the H/P domain by use of proteolytic fragments of rbHPRG and was further confirmed and characterized in two in vivo models of angiogenesis: the chorioallantoic membrane of the chick assay and the mouse Matrigel plug assay. Caspase-3 activation was observed in HUVECs stimulated with FGF-2 in the presence of rbHPRG, suggesting that apoptosis of activated endothelial cells may be one of the mechanisms underlying its antiangiogenic activity. Finally, the H/P domain of rbHPRG reduced tumor cell number when tumor cells were co-inoculated in the Matrigel plug assay. In conclusion, the H/P domain within HPRG induces the apoptosis of activated endothelial cells leading to potent antiangiogenic effects.