Published online before print September 30, 2004, doi: 10.1161/01.RES.0000146674.38319.07
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© 2004 American Heart Association, Inc.
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Platelets Release CXCL4L1, a Nonallelic Variant of the Chemokine Platelet Factor-4/CXCL4 and Potent Inhibitor of Angiogenesis
From the Laboratory of Molecular Immunology (S.S., P.P., J.V.D.), Rega Institute, University of Leuven, Belgium; and Division of Pulmonary and Critical Care Medicine (M.D.B., R.M.S.), David Geffen School of Medicine at University of California Los Angeles.
Correspondence to Jo Van Damme, Laboratory of Molecular Immunology, Rega Institute, University of Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium. E-mail jozef.vandamme{at}rega.kuleuven.ac.be
Abstract
Platelet factor-4 (PF-4)/CXCL4 was the first chemokine described to inhibit neovascularization. Here, the product of the nonallelic variant gene of CXCL4, PF-4var1/PF-4alt, designated CXCL4L1, was isolated for the first time from thrombin-stimulated human platelets and purified to homogeneity. Although secreted CXCL4 and CXCL4L1 differ in only three amino acids, CXCL4L1 was more potent in inhibiting chemotaxis of human microvascular endothelial cells toward interleukin-8 (IL-8)/CXCL8 or basic fibroblast growth factor (bFGF). In vivo, CXCL4L1 was also more effective than CXCL4 in inhibiting bFGF-induced angiogenesis in rat corneas. Thus, activated platelets release CXCL4L1, a potent regulator of endothelial cell biology, which affects angiogenesis and vascular diseases.
Key Words: angiogenesis • endothelial cells • CXCL8/IL-8 • chemokines
The chemokine family consists of proinflammatory cytokines, primarily involved in chemoattraction and activation of specific leukocytes in various immunoinflammatory responses.1–4 In addition, chemokines activate blood platelets, important components of hemostasis, contributing to wound healing by forming thrombi and to the initiation of repair processes.5 However, a recent study indicated that circulating activated platelets and platelet-leukocyte aggregates promote formation of atherosclerotic lesions.6 Finally, chemokines influence tumor growth by regulating angiogenesis. Net angiogenesis is determined by a balance between angiogenic and angiostatic factors within the local microenvironment. The CXC chemokine family is unique because it comprises angiogenic and angiostatic chemokines.7–9
The first chemokine described as a regulator of angiogenesis is platelet factor-4 (PF-4)/CXCL4.10 Although this angiostatic platelet-derived chemokine has been the subject of extensive research as a candidate anticancer drug,11 its nonallelic human gene variant PF-4alt/PF-4var1/SCYB4V1* has not been investigated previously.12,13 In addition to post-translational processing (eg, by proteolytical cleavage),14 gene duplication is another means to increase the biological diversification of the human chemokine family. For example, we identified the chemokine LD78ß/CCL3L1, a nonallelic gene variant of CCL3, as the most potent monocyte chemoattractant.14 More than a decade ago, PF-4alt/PF-4var1, the gene of a nonallelic CXCL4-variant was identified.12,13 The predicted mature proteins differ in only three amino acids located in the COOH terminus (P58
L, K66E, and L67H) in CXCL4 and CXCL4L1, respectively.12,13 This region is involved in CXCL4-heparin interaction.15 Here we describe the isolation of natural CXCL4L1 protein from thrombin-stimulated platelets and demonstrate for the first time that the SCYB4V1 gene is translated into biologically active protein. We characterized this chemokine as a potent inhibitor of angiogenesis, which is more effective than authentic CXCL4.
Materials and Methods
An extended Materials and Methods section can be found in the online data supplement available at http://circres.ahajournals.org.
Purification, Identification, and Measurement of CXCL4L1
For large-scale CXCL4 production, platelets were stimulated with thrombin. The conditioned medium was subsequently purified as described. Pure proteins were identified by NH2-terminal amino acid sequence analysis and by mass spectrometry.16
For the detection of human CXCL4, a classical sandwich ELISA was developed as described.16
Biological Assays
Endothelial cell chemotaxis assays were performed as described.17 The total number of migrated cells was counted in 15 separate high-power fields (HPFs; x400). Specific migration was expressed as the number of endothelial cells that migrated per HPF after subtracting the background (unstimulated control).
In vivo angiogenesis was assessed using the rat cornea micropocket assay as described.17
Results and Discussion
The release of chemokines from the 
-granules of outdated platelet preparations was stimulated with thrombin. The conditioned media were concentrated and subsequently fractionated by heparin-Sepharose affinity chromatography (data not shown). The major peak of CXCL4 immunoreactivity eluted from this column at high salt concentrations (1.6 to 1.8 mol/L NaCl) and contained authentic CXCL4 as determined by mass spectrometry. The CXCL4 immunoreactivity detected in fractions with intermediate heparin affinity (eluting at 1 to 1.4 mol/L NaCl) was further purified by reverse-phase high-performance liquid chromatography (RP-HPLC) and eluted in two separate peaks between 32% and 34% acetonitrile (Figure 1A), both corresponding to pure 8-kDa protein bands on SDS-PAGE (Figure 1B). As deduced from a combination of sequence analysis and mass spectrometry (supplemental Table A and supplemental Figure A, available online at http://circres.ahajournals.org), the RP-HPLC fractions N° 57 to 60 contained authentic CXCL4. The NH2-terminal sequence and detected average Mr of the CXCL4 immunoreactivity (fractions N° 53 to 55) eluting at 32% acetonitrile, corresponded to CXCL4L1.13 Authentic CXCL4 with high affinity for heparin was purified in parallel with CXCL4L1, and homogenous preparations of the mature proteins were compared for angiostatic activity.
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Basic fibroblast growth factor (bFGF; 50 ng/mL) and CXCL8 (80 ng/mL) induced human microvascular endothelial cell (HMVEC) chemotaxis (Figure 2; supplemental Figure B, available online at http://circres.ahajournals.org), which was completely blocked by CXCL4L1 (10 to 100 ng/mL). In contrast, even at 300 ng/mL, authentic CXCL4 failed to inhibit bFGF- or CXCL8-induced chemotaxis. Subsequently, we used the cornea micropocket assay to determine whether CXCL4L1 inhibits angiogenesis in vivo. As shown in Figure 3 (and supplemental Table B, available online at http://circres.ahajournals.org), CXCL4L1 (50 ng) inhibited bFGF (50 ng) in 16 of 22 corneas, representing a 73% inhibition of angiogenesis. In contrast, at the same dose, authentic CXCL4 inhibited bFGF in only 5 of 22 corneas.
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) was compared with CXCL4 (1 to 300 ng/mL; 
) for its capacity to induce migration of HMVECs. In addition, chemotaxis of HMVEC toward bFGF (50 ng/mL) or CXCL8 (80 ng/mL) was inhibited in the presence of different concentrations of CXCL4L1 (
and 
) or CXCL4 (
and 
). To demonstrate specific migration, background (unstimulated control) migration (on average, 38 cells per HPF) was subtracted.
|
The molecular mechanism for CXCL4 angiostatic function is still a matter of debate. It has been suggested that CXCL4 is a unique chemokine that does not bind to a G-protein-coupled receptor but activates cells (ie, neutrophils) and platelets through binding to cell surface glycosaminoglycans (GAG).18,19 The fact that GAG binding is important for CXCL4 interaction is supported by another study demonstrating that CXCL4 and CXCL10 share a GAG binding site on endothelial cells through which these chemokines inhibit FGF-induced endothelial cell growth.20 However, it is not clear whether CXCL4 binding to GAG alone is necessary and sufficient to trigger endothelial cell signaling.21,22 CXCL4 function is not abrogated in heparan sulfate-deficient cells, and CXCL4 mutants or peptides lacking heparin affinity are capable of inhibiting angiogenesis.11 Recently, a splice variant of CXCR3, designated CXCR3B, was identified that binds CXCL4.23 Finally, others have reported that the inhibitory effect of CXCL4 is mediated through complex formation with bFGF or CXCL8.24,25
In this study, we identified CXCL4L1 as another angiostatic factor stored in platelets, which undergo degranulation at sites of vascular injury or thrombosis and modulate net angiogenesis within the local microenvironment. We demonstrated that CXCL4L1, compared with CXCL4, is a 30-fold more potent inhibitor of endothelial cell chemotaxis in vitro and has more profound effects in blocking angiogenesis in vivo. This discovery may have significant implications for the use of CXCL4L1 as a therapeutic tool to inhibit aberrant angiogenesis in a variety of diseases.
Acknowledgments
This work was supported by the Fund for Scientific Research of Flanders (FWO-Vlaanderen), the Concerted Research Actions (GOA) of the Regional Government of Flanders, the Interuniversity Attraction Poles Programme-Belgian Science Policy, the Quality of Life Program of the European Commission, and National Institutes of Health grants CA87879, HL66027, P50CA90388, and P50HL67665. P.P. and S.S. are senior research assistants of the FWO-Vlaanderen. We are grateful for the buffy coats and platelet preparations received from the Red Cross blood transfusion centers of Antwerp and Leuven.
Footnotes
*Accession numbers P10720 and M26167 at Swiss-Prot and Genbank databases for the CXCL4L1 protein and gene, respectively. 
Original received August 20, 2004; revision received September 8, 2004; accepted September 21, 2004.
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