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A short peptide domain of platelet factor 4 blocks angiogenic key events induced by FGF-2¹

MARTIN HAGEDORN^*,3, LIOR ZILBERBERG^*,3, ROSA M. LOZANO, PEDRO CUEVAS, XAVIER CANRON^*, MARIANO REDONDO-HORCAJO, GUILLERMO GIMÉNEZ-GALLEGO and ANDREAS BIKFALVI^*2

^* Growth Factor and Cell Differentiation Laboratory, University Bordeaux I, 33405 Talence, France;
Laboratorio de Estructura y Función de Proteínas. Centro de Investigaciones Biológicas (CSIC); Velázquez, 144. 28006 Madrid, Spain; and
Department of Histology, Hospital Ramón y Cajal, Madrid, Spain

²Correspondence: Growth Factor and Cell Differentiation Laboratory, University Bordeaux I, 33405 Talence, France. E-Mail: a.bikfalvi{at}croissance.u-bordeaux.fr

SPECIFIC AIMS

Identifying small anti-angiogenic peptide fragments that derive from larger proteins of the clotting cascade, basement membranes or the extracellular matrix, may result in the development of lead compounds for the treatment of angiogenesis-dependent diseases such as cancer, diabetic retinopathy, and rheumatoid arthritis. Here we characterize such a peptide from the C-terminus of platelet factor 4 (PF-4), which inhibits endothelial cell proliferation, migration, microvessel assembly, and in vivo suppression of new blood vessel formation at micromolar doses by specifically interfering with FGF-2/FGFR function.

PRINCIPAL FINDINGS

1. PF-4^47–70 inhibits FGF-2 binding to high- and low-affinity receptors, FGF-2 internalization, and FGF-2-dependent cell proliferation
Peptide PF-4^47–70, but not the mutant PF-4^47–70S (C⁵²S), blocked binding of ¹²⁵I-FGF-2 to high- and low-affinity receptors on BCE cells. Concentrations necessary for half-maximal inhibition (IC₅₀) were 2 µM for high-affinity receptors and 4 µM for HSPGs type receptors. Micromolar doses of PF-4^47–70 also inhibited internalization of ¹²⁵I-FGF-2 in BCE cells by 4.6-fold; 20 µM of PF-4^47–70 suppressed ACE cell proliferation below levels of control cells (1% serum), whereas PF-4^47–70S had no significant effect on proliferation at the same concentration. These data suggest a defined interaction of peptide PF-4^47–70 with FGF-2 pathways and a specific role for C⁵² for this effect, although low-resolution structural analysis with circular dichroism spectra shows that the substitution C⁵²S does not modify the secondary structure of PF-4^47–70.

2. Endothelial cell migration and MAPK activation is blocked by PF-4^47–70
PF-4^47–70 strongly inhibited FGF-2-induced BCE migration into the denuded area in a monolayer wounding assay, comparable with cultures after 18 h of serum deprivation without FGF-2 or with cultures treated with the inactive peptide PF-4^47–70S. We therefore investigated whether underlying pro-angiogenic signaling events were affected by PF-4^47–70. Short-term incubation of BCE cells by FGF-2 showed a strong increase in phosphorylation of ERK-isoforms. If co-incubated with 20 µM of peptide PF-4^47–70, ERK1/2 activation decreased markedly, whereas PF-4^47–70S did not influence ERK phosphorylation.

3. Microvessel assembly in aortic ring cultures embedded in collagen gels is hindered in presence of PF-4^47–70
Rat aortic ring cultures incubated in a serum-free collagen matrix give rise to numerous vessel sprouts from the injured edges of the aorta fragment after 4 to 6 days (Fig. 1A , C ). 20 µM of PF-4^47–70 significantly reduced the mean vessel length by 89%, and the vessel number by 86%. The inhibitory effect was dose-dependent, with a narrow concentration range 10–20 µM. PF-4^47–70S did not inhibit formation of vessels; they resembled those of untreated controls (Fig. 1E , F ).

View larger version (44K): [in this window] [in a new window]   Figure 1. Inhibitory effects of PF-447–70 on spontaneous microvascular sprouting in the serum-free rat aortic ring model. Photos from control (A, C) and PF-447–70 treated (B, D) aortic ring cultures. In controls, numerous vessels are visible after 4–6 days. When cultures were treated with 20 µM PF-447–70, vessel formation is strongly inhibited, whereas cellular (fibroblastic) outgrowth is less affected (B, D). If vessels developed, they were much thinner and shorter than vessels in control cultures (B). Magnification x 40 (A, B; bar=1 mm) and x 200 (C, D; bar=130 µm). PF-447–70 reduces vessel length by 89% and vessel number by 86% (E); inhibition is dose-dependent, and peptide PF-447–70S had no effect on vessel growth (F).

4. Systemic treatment with PF-4^47–70 reduced vessel ingrowth in FGF-2-containing subcutaneous sponges in mice
To simulate a more realistic treatment situation, we investigated the effects of systemic delivery of PF-4^47–70 on angiogenesis in a gelatin sponge assay. A single intraperitoneal injection of PF4^47–70 24 h after implantation decreased angiogenesis by 86% (calculating areas containing erythrocytes, Fig. 2F , G ) or by 81% (counting laminin-positive vessels, Fig. 2D , G ). No new capillaries were found in sponges incubated with PBS alone or with peptide PF-4^47–70 in PBS (Fig. 2A , B ), whereas sponges containing 10 ng/ml FGF-2 were infiltrated by new blood vessels (Fig. 2C , E ). PF-4^47–70-treated vessels were scattered and of immature nature, and they had smaller diameters; whereas slightly more inflammatory cells were present (Fig. 2D , F ).

View larger version (86K): [in this window] [in a new window]   Figure 2. Inhibition of in vivo angiogenesis by PF447–70 in the mouse gelatin sponge assay. Photos (original magnification x 40) of sponges preincubated with 200 µl of PBS alone (A), 45 µg/ml of PF447–70 in PBS (B), 10 ng/ml FGF-2 in PBS plus an injection of PBS 24 h after surgery (C, E), or 10 ng/ml FGF-2 plus one injection of 16 µM PF447–70 (D, F). Note the absence of neovessels in sponges embedded with PBS or PF447–70 alone (A, B). Arrows indicate newly formed vessels, counterstaining by hematoxylin (A–D) or hematoxylin and eosin (E, F). Quantification of the angiogenesis was performed either by calculating the area covered by erythrocytes or by counting laminin-positive vessels (G).

CONCLUSION

Full-length PF-4 has been shown to be a potent angiogenesis inhibitor in various in vitro and in vivo models. Anti-angiogenic therapy may require long-term treatment with protein drugs; it is necessary to identify smaller portions of natural inhibitors to reduce production costs and immunogenicity. For this purpose, we have characterized a 24-amino-acid C-terminal fragment of PF-4 (PF-4^47–70). Its anti-angiogenic features are based on the defined interaction with FGF-2 and its receptors. ¹²⁵I-FGF-2-binding to high- and low-affinity receptors on the surface of BCE cells is strongly reduced by PF-4^47–70 but not by the C⁵²S mutant PF-4^47–70S. Inhibition of FGF-2 binding is a mode of action also shared by other anti-angiogenic molecules like TSP-1, PD 166866 and RG-13577. Further activities of endothelial cells require signaling by MAPK members ERK1/2. PF-4^47–70 reduces FGF-2-triggered activation of MAPK ERK1/2 as it has been shown for angiostatin, a potent angiogenesis inhibitor whose overall mechanism of action is not yet fully understood. ERK activation by growth factor receptors (e.g., FGFR) or integrins plays an important role in cell motility of endothelial cells and other cell types. Endogenous angiogenesis inhibitors like the 16-kDa fragment of prolactin or angiostatin can block FGF-2-induced phosphorylation of ERK isoforms p42/p44 in vitro. PF-4^47–70 strongly reduces endothelial cell migration in a monolayer wounding assay. Others have found that inhibition of migration might be linked to reduced ERK-1/2 phosphorylation, because PD 98059 a specific blocker of ERK, inhibits cell migration in this assay. Further assembly of endothelial cells into a functional vessel requires chord organization and lumen formation, which can be studied in the serum-free aortic ring assay. PF-4^47–70 inhibits almost completely microvessel outgrowth in this assay, but affects stroma cell proliferation less efficiently (Fig. 1) . These data indicate that PF-4^47–70 acts as a suppressor of the first steps of microvessel maturation. Recently, murine endostatin has been evaluated by using this method and was found to block vessel sprouting at 9-fold higher concentrations as PF-4^47–70.

In vivo blood vessel growth was studied in the mouse sponge assay. A single injection of PF-4^47–70 is sufficient to strongly suppress the angiogenic response in FGF-2 containing sponges compared with control mice (Fig. 2C , D , E , F , G ). The N-terminal NGR sequence of PF-4^47–70 may enhance biological effects in vivo by targeting the peptide to sites of active angiogenesis. This finding would be in accordance with the work of Borgstrom and co-workers, who found that recombinant PF-4 accumulates at sites of active angiogenesis, and of Arap et al., who identified the NGR peptide motif as a neovascular homing domain. Note that PF-4^47–70S is devoid of any anti-angiogenic activity, which indicates that a free cysteine (C⁵²) must be present for angiogenesis inhibition. Taken together, PF-4^47–70 regroups most of the important features of a potent angiogenesis inhibitor (Fig. 3 ). Further studies should investigate its effects on vessel maturation and arteriogenesis. Because of its small size, its known mode of action and its defined structure, PF-4^47–70 is a very promising candidate for further development as an anti-angiogenic drug for the treatment of cancer and other angiogenesis-dependent diseases.

View larger version (66K): [in this window] [in a new window]   Figure 3. Diagram illustrating the cellular events resulting from the interaction of PF-447–70 with FGF-2. PF-447–70 consists of 24 amino acids compared with the tetrameric human PF-4 (280 amino acids, image generated with Cn3D v3.0). It associates with FGF-2 and leads to a conformation change of the growth factor. Alteration of the secondary structure impairs FGF-2 dimerization and inhibits binding to tyrosine kinase receptors (high-affinity receptors) and to cell surface proteoglycans (low-affinity receptors, not drawn). A proper binding of the FGF-2 molecule is further necessary for receptor dimerization and internalization, and for transducing a pro-angiogenic growth signal into the cell. Consequently, blocking of FGF-2/FGFR-interaction leads to down-regulation of MAPK phosphorylation, a major downstream signaling pathway. Biological consequences of these interactions are inhibition of endothelial cell proliferation, migration, microvessel assembly, and in vivo angiogenesis.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0285fje ; to cite this article, use FASEB J. (January 5, 2001) 10.1096/fj.00-0285fje

³ These authors contributed equally to this paper.

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