Platelet Factor-4 Inhibits the Mitogenic Activity of VEGF121 and VEGF165 Using Several Concurrent Mechanisms

Abstract

The 121-amino acid form of vascular endothelial growth factor (VEGF121) and the 165-amino acid form (VEGF165) are mitogenic for vascular endothelial cells and induce angiogenesis in vivo. VEGF165 possesses a heparin binding ability and in the absence of heparin-like molecules does not bind efficiently to the VEGF receptors of vascular endothelial cells. The binding of 125I-VEGF165 to the VEGF receptors of endothelial cells, and the heparin-dependent binding of 125I-VEGF165 to a soluble extracellular domain of the VEGF receptor KDR/flk-1, were inhibited by the angiogenesis inhibitor platelet factor-4 (PF4). In contrast, PF4 was not able to inhibit the binding of VEGF121, a VEGF isoform which lacks a heparin binding capacity, to the VEGF receptors of the cells or to KDR/flk-1. These results indicate that PF4 may inhibit VEGF165 binding to VEGF receptors by disrupting the interaction of VEGF165 with cell surface heparan sulfates. Since PF4 mutants lacking a heparin binding ability retain their anti-angiogenic activity, alternative inhibitory mechanisms were also examined. 125I-PF4 bound with high affinity (Kd 5 x 10(-9) M) to VEGF165-coated wells. The binding of 125I-PF4 to the VEGF165-coated wells was inhibited by several types of heparin binding proteins, including unlabeled PF4 and unlabeled VEGF165. The binding was not inhibited by proteins which lack a heparin binding capacity, nor was it inhibited by VEGF121. Heparinase did not inhibit the binding of 125I-PF4 to VEGF165, indicating that heparin-like molecules are not required. These experiments suggest that PF4 can bind to heparin binding proteins such as VEGF165 leading to an inhibition of their receptor binding ability. In agreement with these results, we have observed that PF4 inhibits efficiently the VEGF165 induced proliferation of vascular endothelial cells. Unexpectedly, PF4 also inhibited efficiently the VEGF121-induced proliferation of the cells, indicating that PF4 can disrupt VEGF receptor mediated signal transduction using an unknown mechanism which does not interfere with VEGF121 binding.

The COOH-Terminal Peptide of Platelet Factor-4 Variant (CXCL4L1/PF-4var47-70) Strongly Inhibits Angiogenesis and Suppresses B16 Melanoma Growth In vivo

“…Further, it has been shown that different CXCL4/PF-4 peptides compete with FGF-2 or VEGF by inhibiting their binding to FGF or VEGF receptors, respectively, or reduce heparin-induced FGF-2 dimerization, resulting in the angiostatic activity (21,31,44,45). However, other mechanisms seem to be involved as well because an analogue of CXCL4/PF-4 that lacks affinity for heparin still retains its angiostatic activity (46), and CXCL4/PF-4 also inhibits the function of VEGF-121, an isoform with deficient heparin-binding ability (47). Recently, an interaction of CXCL4/PF-4 with integrins has been described that might be implicated in angiogenesis (48).…”

The VEGF angiogenic switch of fibroblasts is regulated by MMP-7 from cancer cells

“…Previous reports have shown that some secreted proteins, including soluble VEGF receptor 1, platelet factor 4, thrombospondin, ADAMTS1 and CTGF, bind to VEGF and inhibit angiogenic activity (Kendall and Thomas, 1993;Gengrinovitch et al, 1995;Gupta et al, 1999;Luque et al, 2003;Inoki et al, 2002;Jang et al, 2004). As one of these proteins, CTGF is typically expressed in fibroblasts (Brigstock, 1999), we considered the possibility that CTGF might function as an inhibitor of VEGF from fibroblasts.…”

“…However, the mechanism by which vascular quiescence is maintained in normal tissues, wherein VEGF is widely expressed (Berse et al, 1992;Hanahan and Folkman, 1996), remains unclear. Several secreted proteins have been reported to bind and sequester VEGF (Kendall and Thomas, 1993;Gengrinovitch et al, 1995;Gupta et al, 1999;Inoki et al, 2002;Luque et al, 2003). This type of regulation could account for the vascular quiescence in the presence of VEGF, although in most tissues except the cornea (Ambati et al, 2006), the relevance of these proteins to vascular control has yet to be demonstrated.…”

Platelet Factor 4/CXCL4 Induces Phagocytosis and the Generation of Reactive Oxygen Metabolites in Mononuclear Phagocytes Independently of Gi Protein Activation or Intracellular Calcium Transients

“…Although being structurally a chemokine, PF-4 lacks chemotactic activity on neutrophils and T cells (15,16). Apart from inducing short term responses on PMN, PF-4 is involved in long term differentiation and regulatory processes such as the control of EC and fibroblast proliferation (17)(18)(19) and the support of the survival of hemopoietic stem cells and progenitor cells (20). Moreover, PF-4 suppresses IL-2 mRNA transcription and the release of IL-2 in activated human T cells, corresponding to its inhibitory effect on T cell proliferation and release of IFN-␥ (15).…”