Angiostatic and chemotactic activities of the CXC chemokine CXCL4L1 (platelet factor-4 variant) are mediated by CXCR3.

We investigated possible cellular receptors for the human CXC chemokine platelet factor-4 variant/CXCL4L1, a potent inhibitor of angiogenesis. We found that CXCL4L1 has lower affinity for heparin and chondroitin sulfate-E than platelet factor-4 (CXCL4) and showed that CXCL10 and CXCL4L1 could displace each other on microvascular endothelial cells. Labeled CXCL4L1 also bound to CXCR3A- and CXCR3B-transfectants and was displaced by CXCL4L1, CXCL4, and CXCL10. The CXCL4L1 anti-angiogenic activity was blocked by anti-CXCR3 antibodies (Abs) in the Matrigel and cornea micropocket assays. CXCL4L1 application in CXCR3(-/-) or in wild-type mice treated with neutralizing anti-CXCR3 Abs, resulted in reduced inhibitory activity of CXCL4L1 on tumor growth and vascularization of Lewis lung carcinoma. Furthermore, CXCL4L1 and CXCL4 chemoattracted activated T cells, human natural killer cells, and human immature dendritic cells (DCs). Migration of DCs toward CXCL4 and CXCL4L1 was desensitized by preincubation with CXCL10 and CXCL11, inhibited by pertussis toxin, and neutralized by anti-CXCR3 Abs. Chemotaxis of T cells, natural killer cells, and DCs is likely to contribute to the antitumoral action. However, the in vivo data indicate that the angiostatic property of CXCL4L1 is equally important in retarding tumor growth. Thus, both CXCR3A and CXCR3B are implicated in the chemotactic and vascular effects of CXCL4L1.

The role of the CXC chemokines platelet factor-4 (CXCL4/PF-4) and its variant (CXCL4L1/PF-4var) in inflammation, angiogenesis and cancer.

Chemokines are chemotactic cytokines which recruit leukocytes to inflammatory sites. They also affect tumor development and metastasis by acting as growth factor, by attracting pro- or anti-tumoral leukocytes or by influencing angiogenesis. Platelet factor-4 (CXCL4/PF-4) was the first chemokine shown to inhibit angiogenesis. CXCL4L1/PF-4var, recently isolated from thrombin-stimulated platelets, differing from authentic CXCL4/PF-4 in three carboxy-terminally located amino acids, was found to be more potent than CXCL4/PF-4 in inhibiting angiogenesis and tumor growth. Both glycosaminoglycans (GAG) and CXCR3 are implicated in the activities of the PF-4 variants. This report reviews the current knowledge on the role of CXCL4/PF-4 and CXCL4L1/PF-4var in physiological and pathological processes. In particular, the role of CXCL4/PF-4 in cancer, heparin-induced thrombocytopenia and atherosclerosis is described.

The COOH-terminal peptide of platelet factor-4 variant (CXCL4L1/PF-4var47-70) strongly inhibits angiogenesis and suppresses B16 melanoma growth in vivo.

Chemokines influence tumor growth directly or indirectly via both angiogenesis and tumor-leukocyte interactions. Platelet factor-4 (CXCL4/PF-4), which is released from alpha-granules of activated platelets, is the first described angiostatic chemokine. Recently, it was found that the variant of CXCL4/PF-4 (CXCL4L1/PF-4var) could exert a more pronounced angiostatic and antitumoral effect than CXCL4/PF-4. However, the molecular mechanisms of the angiostatic activities of the PF-4 forms remain partially elusive. Here, we studied the biological properties of the chemically synthesized COOH-terminal peptides of CXCL4/PF-4 (CXCL4/PF-4(47-70)) and CXCL4L1/PF-4var (CXCL4L1/PF-4var(47-70)). Both PF-4 peptides lacked monocyte and lymphocyte chemotactic activity but equally well inhibited (25 nmol/L) endothelial cell motility and proliferation in the presence of a single stimulus (i.e., exogenous recombinant fibroblast growth factor-2). In contrast, when assayed in more complex angiogenesis test systems characterized by the presence of multiple mediators, including in vitro wound-healing (2.5 nmol/L versus 12.5 nmol/L), Matrigel (60 nmol/L versus 300 nmol/L), and chorioallantoic membrane assays, CXCL4L1/PF-4var(47-70) was found to be significantly (5-fold) more angiostatic than CXCL4/PF-4(47-70). In addition, low (7 microg total) doses of intratumoral CXCL4L1/PF-4var(47-70) inhibited B16 melanoma growth in mice more extensively than CXCL4/PF-4(47-70). This antitumoral activity was predominantly mediated through inhibition of angiogenesis (without affecting blood vessel stability) and induction of apoptosis, as evidenced by immunohistochemical and fluorescent staining of B16 tumor tissue. In conclusion, CXCL4L1/PF-4var(47-70) is a potent antitumoral and antiangiogenic peptide. These results may represent the basis for the design of CXCL4L1/PF-4var COOH-terminal-derived peptidomimetic anticancer drugs.

Expression of angiostatic platelet factor-4var/CXCL4L1 counterbalances angiogenic impulses of vascular endothelial growth factor, interleukin-8/CXCL8, and stromal cell-derived factor 1/CXCL12 in esophageal and colorectal cancer.

Chemokines influence tumor progression through regulation of leukocyte chemotaxis, angiogenesis, and metastasis. In this study, the regulated expression of angiogenic (stromal cell-derived factor [SDF]-1/CXCL12 and interleukin [IL]-8/CXCL8) and angiostatic (platelet factor [PF]-4var/CXCL4L1 and inducible protein [IP-10]/CXCL10) chemokines was examined in human colorectal and esophageal cancer. In HCT 116 and HCT-8 colorectal adenocarcinoma cells, the production of IL-8 immunoreactivity was up-regulated by IL-1beta, tumor necrosis factor (TNF)-alpha, the toll-like receptor (TLR) ligands double-stranded RNA and peptidoglycan and phorbol ester. Increased PF-4 and synergistic IL-8 and IP-10 induction in carcinoma cells after stimulation with IL-1beta plus TNF-alpha or interferon-gamma was demonstrated by enzyme-linked immunosorbent assay, quantitative reverse transcriptase polymerase chain reaction, or immunocytochemistry. In addition, IL-8 from HT-29 colorectal adenocarcinoma cells was molecularly identified as intact chemokine, as well as NH(2)-terminally truncated, more active IL-8(6-77). The presence of PF-4var, SDF-1, and vascular endothelial growth factor (VEGF) was evidenced by immunohistochemistry in surgical samples from 51 patients operated on for colon adenocarcinoma (AC), esophageal AC, or esophageal squamous cell carcinoma (SCC). PF-4var was strongly detected in colorectal cancer, whereas its expression in esophageal cancer was rather weak. Staining for SDF-1 was almost negative in esophageal SCC, whereas a more intense and frequent staining was observed in AC of the esophagus and colon. Staining for VEGF was moderately to strongly positive in all 3 types of cancer, although less prominent in esophageal AC. The heterogenous expression of angiogenic (IL-8, SDF-1) as well as angiostatic (IP-10, PF-4var) chemokines not only within the tumor and between the different cases but also between the different tumor cell types may indicate a distinct role of the various chemokines in the complex process of tumor development.

Synergistic up-regulation of MCP-2/CCL8 activity is counteracted by chemokine cleavage, limiting its inflammatory and anti-tumoral effects.

Chemokines mediate the inflammatory response by attracting various leukocyte types. MCP-2/CC chemokine ligand 8 (CCL8) was induced at only suboptimal levels in fibroblasts and endothelial cells by IL-1beta or IFN-gamma, unless these cytokines were combined. IFN-gamma also synergized with the TLR ligands peptidoglycan (TLR2), dsRNA (TLR3) or LPS (TLR4). Under these conditions, intact MCP-2/CCL8(1-76) produced by fibroblasts was found to be processed into MCP-2/CCL8(6-75), which lacked chemotactic activity for monocytic cells. Furthermore, the capacity of MCP-2/CCL8(6-75) to increase intracellular calcium levels through CCR1, CCR2, CCR3 and CCR5 was severely reduced. However, the truncated isoform still blocked these receptors for other ligands. MCP-2/CCL8(6-75) induced internalization of CCR2, inhibited MCP-1/CCL2 and MCP-2/CCL8 ERK signaling and antagonized the chemotactic activity of several CCR2 ligands (MCP-1/CCL2, MCP-2/CCL8, MCP-3/CCL7). In contrast to MCP-3/CCL7, parvoviral delivery of MCP-2/CCL8 into B78/H1 melanoma failed to inhibit tumor growth, partially due to proteolytic cleavage into inactive MCP-2/CCL8 missing five NH(2)-terminal residues. However, in an alternative tumor model, using HeLa cells, MCP-2/CCL8 retarded tumor development. These data indicate that optimal induction and delivery of MCP-2/CCL8 is counteracted by converting this chemokine into a receptor antagonist, thereby losing its anti-tumoral potential.

The role of CXC chemokines and their receptors in cancer.

Chemokines, or chemotactic cytokines, and their receptors have been discovered as essential and selective mediators in leukocyte migration to inflammatory sites and to secondary lymphoid organs. Besides their functions in the immune system, they also play a critical role in tumor initiation, promotion and progression. There are four subgroups of chemokines: CXC, CC, CX(3)C, and C chemokine ligands. The CXC or alpha subgroup is further subdivided in the ELR(+) and ELR(-) chemokines. Members that contain the ELR motif bind to CXC chemokine receptor 2 (CXCR2) and are angiogenic. In contrast, most of the CXC chemokines without ELR motif bind to CXCR3 and are angiostatic. An exception is the angiogenic ELR(-)CXC chemokine stromal cell-derived factor-1 (CXCL12/SDF-1), which binds to CXCR4 and CXCR7 and is implicated in tumor metastasis. This review is focusing on the role of CXC chemokines and their receptors in tumorigenesis, including angiogenesis, attraction of leukocytes to tumor sites and induction of tumor cell migration and homing in metastatic sites. Finally, their therapeutic use in cancer treatment is discussed.

Stimulation of angiostatic platelet factor-4 variant (CXCL4L1/PF-4var) versus inhibition of angiogenic granulocyte chemotactic protein-2 (CXCL6/GCP-2) in normal and tumoral mesenchymal cells.

Chemokines affect inflammation and cancer through leukocyte attraction and angiogenesis. Here, we demonstrate that CXCL4L1/platelet factor-4 variant (PF-4var), a highly angiostatic chemokine, is poorly chemotactic for phagocytes and is inducible in monocytes by inflammatory mediators but remained undetectable in macrophages and neutrophils. In addition, CXCL4L1/PF-4var production by mesenchymal tumor cells was evidenced in vitro and in vivo by specific ELISA and immunohistochemistry. CXCL4L1/PF-4var, but not CXCL4/PF-4, was coinduced with the angiogenic chemokine CXCL6/granulocyte chemotactic protein-2 (GCP-2) by cytokines, e.g., IL-1beta and IL-17, in sarcoma cells, but not in diploid fibroblasts. Furthermore, the induction of CXCL6/GCP-2 in endothelial cells by IL-1beta was enhanced synergistically by TNF-alpha but inhibited by IFN-gamma, which synergized with IL-1beta to produce the angiostatic CXCL10/IFN-gamma-induced protein-10. These findings indicate that the equilibrium between angiostatic and angiogenic factors during inflammation and tumor progression is rather complex and differs depending on the chemokine, cell type, and stimulus. Selective intervention in the chemokine network may drastically disturb this delicate balance of angiogenesis and tissue repair. Application of angiostatic CXCL4L1/PF-4var without attraction of protumoral phagocytes may be beneficial in cancer therapy.

Proteolytic processing of CXCL11 by CD13/aminopeptidase N impairs CXCR3 and CXCR7 binding and signaling and reduces lymphocyte and endothelial cell migration.

CXCR3 ligands were secreted by tissue fibroblasts and peripheral blood-derived mononuclear leukocytes in response to interferon-gamma (IFN-gamma) and Toll-like receptor (TLR) ligands. Subsequent purification and identification revealed the presence of truncated CXCL11 variants missing up to 6 amino acids. In combination with CD26/dipeptidyl peptidase IV, the metalloprotease aminopeptidase N (APN), identical to the myeloid cell marker CD13, rapidly processed CXCL11, but not CXCL8, to generate truncated CXCL11 forms. Truncated CXCL11 had reduced binding, signaling, and chemotactic properties for lymphocytes and CXCR3- or CXCR7-transfected cells. CD13/APN-truncated CXCL11 failed to induce an intracellular calcium increase but was still able to bind and desensitize CXCR3 for intact CXCL11 signaling. CXCL11 efficiently bound to CXCR7, but CXCL11 was not able to induce calcium signaling or ERK1/2 or Akt phosphorylation through CXCR7. CD26-truncated CXCL11 failed to attract lymphocytes but still inhibited microvascular endothelial cell (HMVEC) migration. However, further processing of CXCL11 by CD13 resulted in significant reduction of inhibition of HMVEC migration. Taken together, during inflammation or cancer, CXCL11 processing by CD13 may lead to a reduced number of tumor-infiltrating lymphocytes and in a more angiogenic environment.