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.     

Identification of the platelet-derived chemokine CXCL4/PF-4 as a broad-spectrum HIV-1 inhibitor

The natural history of HIV-1 infection is highly variable in different individuals, spanning from a rapidly progressive course to a long-term asymptomatic infection. A major determinant of the pace of disease progression is the in vivo level of HIV-1 replication, which is regulated by a complex network of cytokines and chemokines expressed by immune and inflammatory cells. The chemokine system is critically involved in the control of HIV-1 replication by virtue of the role played by specific chemokine receptors, most notably CCR5 and CXCR4, as cell-surface coreceptors for HIV-1 entry; hence, the chemokines that naturally bind such coreceptors act as endogenous inhibitors of HIV-1. Here, we show that the CXC chemokine CXCL4 (PF-4), the most abundant protein contained within the α-granules of platelets, is a broad-spectrum inhibitor of HIV-1 infection. Unlike other known HIV-suppressive chemokines, CXCL4 inhibits infection by the majority of primary HIV-1 isolates regardless of their coreceptor-usage phenotype or genetic subtype. Consistent with the lack of viral phenotype specificity, blockade of HIV-1 infection occurs at the level of virus attachment and entry via a unique mechanism that involves direct interaction of CXCL4 with the major viral envelope glycoprotein, gp120. The binding site for CXCL4 was mapped to a region of the gp120 outer domain proximal to the CD4-binding site. The identification of a platelet-derived chemokine as an endogenous antiviral factor may have relevance for the pathogenesis and treatment of HIV-1 infection.     

The stromal cell-derived factor-1 chemokine is a potent platelet agonist highly expressed in atherosclerotic plaques

Chemokines are chemotactic cytokines that activate and direct the migration of leukocytes. However, their role in modulating platelet function has not been shown. We studied the direct effect of chemokines on human platelets and found that of the 16 tested only stromal cell-derived factor (SDF)-1 induced platelet aggregation, accompanied by a rise in intracellular calcium. Platelets expressed the SDF-1 receptor, CXCR4, and an antibody to CXCR4 and pertussis toxin inhibited SDF-1-induced platelet aggregation, confirming that this effect is mediated through CXCR4, a Galphai-coupled receptor. SDF-1-induced platelet aggregation was also inhibited by wortmannin, LY294002, and genistein, suggesting that phosphatidylinositol 3-kinase and tyrosine kinase are likely involved in SDF-1-induced platelet aggregation. Because chemokines are produced from multiple vascular cells and atherosclerotic vessels are prone to develop platelet-rich thrombi, we examined the expression of SDF-1 in human atheroma. SDF-1 protein was highly expressed in smooth muscle cells, endothelial cells, and macrophages in human atherosclerotic plaques but not in normal vessels. Our studies demonstrate a direct effect of a chemokine in inducing platelet activation and suggest a role for SDF-1 in the pathogenesis of atherosclerosis and thrombo-occlusive diseases.     

The control of angiogenesis and tumor invasion by platelet factor-4 and platelet factor-4-derived molecules

Platelet factor-4 (PF-4) is an ELR-negative chemokine that exhibits antiangiogenesis properties. PF-4 inhibits endothelial cell proliferation and migration, angiogenesis in vitro and in vivo, and tumor growth. However, tumor cells are not directly inhibited by PF-4. To date, a cell surface receptor that would explain the biological effects mediated by PF-4 has not been identified. PF-4 is able to interact directly with angiogenesis growth factors such as fibroblast growth factors (FGFs) and vascular endothelial growth factors and inhibits their interaction with cell surface receptors. Furthermore, dimerization of fibroblast growth factors is abrogated by PF-4. Whether PF-4 plays a role as an endogenous angiogenesis regulator is at present not clear. Several PF-4 fragments and modified molecules have been made that exhibit antiangiogenesis properties. Among these is a C-terminal fragment that has a defined structure, retains all the antiangiogenesis properties of the parent molecule, inhibits growth factor receptor binding, associates with FGFs, and destabilizes their three-dimensional structure. The relevance of these observations for the treatment of malignant disease is discussed.     

The stromal derived factor-1/CXCL12-CXC chemokine receptor 4 biological axis in non-small cell lung cancer metastases

Non-small cell lung cancer is characterized by a specific metastatic pattern. The mechanism for organ-specific metastasis is poorly understood, although evidence has suggested that the chemokine stromal derived factor-1 (CXCL12) and its cognate receptor CXCR4 may regulate breast cancer metastasis. We hypothesized that the CXCL12-CXCR4 biological axis is important in mediating non-small cell lung cancer metastases. Our results indicate that both non-small cell lung cancer tumor specimens resected from patients and non-small cell lung cancer cell lines express CXCR4, but not CXCL12. Non-small cell lung cancer cell lines undergo chemotaxis in response to CXCL12. CXCL12-CXCR4 activation of non-small cell lung cancer cell lines showed intracellular calcium mobilization and mitogen-activated protein kinase activation with enhanced extracellular signal-related kinase-1/2 phosphorylation without change in either proliferation or apoptosis. Target organs in a murine model that are the preferred destination of human non-small cell lung cancer metastases elaborate higher levels of CXCL12 than does the primary tumor, and suggest the generation of chemotactic gradients. The administration of specific neutralizing anti-CXCL12 antibodies to severe combined immunodeficient mice expressing human non-small cell lung cancer abrogated organ metastases, without affecting primary tumor-derived angiogenesis. These data suggest that the CXCL12-CXCR4 biological axis is involved in regulating the metastasis of non-small cell lung cancer.     

PF-4/CXCL4 and CXCL4L1 exhibit distinct subcellular localization and a differentially regulated mechanism of secretion

PF-4/CXCL4 is a member of the CXC chemokine family, which is mainly produced by platelets and known for its pleiotropic biological functions. Recently, the proteic product of a nonallelic variant gene of CXCL4 was isolated from human platelets and named as CXCL4L1. CXCL4L1 shows only 4.3% amino acid divergence in the mature protein, but exhibits a 38% amino acid divergence in the signal peptide region. We hypothesized that this may imply a difference in the cell type in which CXCL4L1 is expressed or a difference in its mode of secretion. In different types of transfected cells, CXCL4 and CXCL4L1 exhibited a distinct subcellular localization and a differential regulation of secretion, CXCL4 being stored in secretory granules and released in response to protein kinase C activation, whereas CXCL4L1 was continuously synthesized and secreted through a constitutive pathway. A protein kinase C-regulated CXCL4 secretion was observed also in lymphocytes, a cell type expressing mainly CXCL4 mRNA, whereas smooth muscle cells, which preferentially expressed CXCL4L1, exhibited a constitutive pathway of secretion. These results demonstrate that CXCL4 and CXCL4L1 exhibit a distinct subcellular localization and are secreted in a differentially regulated manner, suggesting distinct roles in inflammatory or homeostatic processes.     

A potent inhibitor of endothelial cell proliferation is generated by proteolytic cleavage of the chemokine platelet factor 4.

Platelet factor 4 (PF-4) is an archetype of the "chemokine" family of low molecular weight proteins that play an important role in injury responses and inflammation. From activated human leukocyte culture supernatants, we have isolated a form of PF-4 that acts as a potent inhibitor of endothelial cell proliferation. The PF-4 derivative is generated by peptide bond cleavage between Thr-16 and Ser-17, a site located downstream from the highly conserved and structurally important CXC motif. The unique cleavage leads to a loss of one of the structurally important large loops in the PF-4 molecule and generation of an N terminus with basic residues that have the potential to interact with the acidic extracellular domain of the G-protein-coupled chemokine receptor. The N-terminal processed PF-4 exhibited a 30- to 50-fold greater growth inhibitory activity on endothelial cells than PF-4. Since endothelial cell growth inhibition is the only known cellular activity of the cleaved PF-4, we have designated this chemokine endothelial cell growth inhibitor. The N-terminal processing of PF-4 may represent an important mechanism for modulating PF-4 activity on endothelial cells during tissue injury, inflammation, and neoplasia.     

The chemokine SDF-1/CXCL12 modulates the firing pattern of vasopressin neurons and counteracts induced vasopressin release through CXCR4

Chemokines play a key role in inflammation. They are expressed not only in neuroinflammatory conditions, but also constitutively by different cell types, including neurons in the normal brain, suggesting that they may act as modulators of neuronal functions. Here, we investigated a possible neuroendocrine role of the chemokine stromal cell-derived factor 1 (SDF-1)/CXCL12. We demonstrated the colocalization of SDF-1 and its receptor CXCR4 with arginine vasopressin (AVP) in the magnocellular neurons of the supraoptic nucleus (SON) and the paraventricular hypothalamic nucleus and on AVP projections to the neurohypophysis. Electrophysiological recordings of SON neurons demonstrated that SDF-1 affects the electrical activity of AVP neurons through CXCR4, resulting in changes in AVP release. We observed that SDF-1 can blunt the autoregulation of AVP release in vitro and counteract angiotensin II-induced plasma AVP release in vivo. Furthermore, a short-term physiological increase in AVP release induced by enhanced plasma osmolarity, which was produced by the administration of 1 M NaCl i.p., was similarly blocked by central injection of SDF-1 through CXCR4. A change in water balance by long-term salt loading induced a decrease in both SDF-1 and CXCR4 parallel to that of AVP immunostaining in SON. From these data, we demonstrate that chemokine actions in the brain are not restricted to inflammatory processes. We propose to add to the known autoregulation of AVP on its own neurons, a second autocrine system induced by SDF-1 able to modulate central AVP neuronal activity and release.     

基质细胞衍生因子-1/CXCR4轴与肝脏疾病

基质细胞衍生因子-1(SDF-1)/CXCR4轴及其介导的细胞信号转导通路在肝脏疾病中的作用是国内外研究的热点.研究发现SDF-1/ CXCR4信号转导途径与肝脏再生、炎症、肝硬化以及肿瘤等疾病有关,但其具体机制尚未完全清楚.在细胞微环境中,SDF-1/CXCR4相互作用促进肝癌细胞生长,增强肿瘤的迁移、浸润以及转移能力.本文就SDF-1/CXCR4通路在肝再生、炎症、肿瘤疾病中的病理特征和致病机制的研究进展作一综述.     

Human platelet antigen-1a antibodies induce the release of the chemokine RANTES from human platelets

BACKGROUND AND OBJECTIVE: Binding of human platelet antigen-1a (HPA-1a)-specific antibodies to target platelets can trigger platelet activation and mediator release. Here we tested the effect of HPA-1a antibody-containing sera on platelet release of the chemokine RANTES (regulated on activation, normal, T-cell expressed, and presumably secreted) in vitro. PATIENTS AND METHODS: HPA-1a-containing sera obtained from 11 mothers delivered of an infant with neonatal alloimmune thrombocytopenia (NAIT) and from six patients with post-transfusion purpura (PTP) were incubated with HPA-1a/a target platelets. Antibody-induced release of soluble RANTES was determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: A significant release of soluble RANTES was induced by four out of the 17 sera. Two out of the four reactive sera were obtained from mothers who were delivered of a baby with NAIT and the remaining two sera were from patients with PTP. Chemokine release was specific for binding of anti-HPA-1a to the platelet membrane, as none of the reactive sera induced the release of soluble RANTES when incubated with HPA-1b/b platelets. The blockade of platelet-expressed Fc gamma receptor type II (FcgammaRII) inhibited anti-HPA-1a-mediated RANTES release when incubated with the reactive sera of patients with NAIT, but not when platelets were incubated with sera of patients with PTP. CONCLUSION: Our findings suggest that anti-HPA-1a antibody-induced release of platelet-derived RANTES can play a role in adverse reactions in alloimmunized patients.     

SDF-1/CXCR4轴在肝脏干细胞移植中的应用

基质细胞衍生因子-1(stromal cell-derivedfactor-1, SDF-1)及其受体CXCR4构成SDF-1/CXCR4轴体系统.近年来随着该领域科研的不断深入, 越来越多的研究发现SDF-1/CXCR4轴体系统在组织损伤及修复中起着重要的作用.本文主要对SDF-1/CXCR4轴体系统的生物学特性及其在肝脏干细胞移植中的作用进行综述.     

[Phe4]somatostatin: a potent, selective inhibitor of growth hormone release.

[Phe4]Somatostatin was twice as active as somatostatin (SS) in suppressing rat growth hormone release in vitro but had only weak activity toward inhibition of insulin and glucagon release in vivo. The ability of this analogue to inhibit growth hormone release more actively than SS was confirmed in vivo by two separately designed bioassays. Further structure/activity studies of position 4 were carried out with [Glu4]SS, [Thr4]SS, and des-Lys4-SS, all of which had negligible inhibiting activity in the pituitary and pancreas. In this context the strikingly selective activity of [Phe4]SS suggests a fundamental difference in the SS receptors of pituitary and pancreas and the normal side-chain basicity of position 4 appears to be more important for action in pancreas than in pituitary. [Phe4]SS has properties that may be useful in the development of agents for the treatment of acromegaly or other disorders associated with increased growth hormone levels.     

Podocytes produce homeostatic chemokine stromal cell-derived factor-1/CXCL12, which contributes to glomerulosclerosis, podocyte loss and albuminuria in a mouse model of type 2 diabetes

Aims/hypothesis  

Chemokine (C-X-C motif) ligand 12 (CXCL12) (also known as stromal cell-derived factor-1 [SDF-1]-alpha) is a homeostatic chemokine with multiple roles in cell homing, tumour metastasis, angiogenesis and tissue regeneration after acute injuries. However, its role in chronic diseases remains poorly defined, e.g. in chronic glomerular diseases like diabetic glomerulosclerosis. We hypothesised that CXCL12 may have a functional role during the evolution of diabetic glomerulosclerosis, either by assisting glomerular repair or by supporting the maladaptive tissue remodelling in response to hyperglycaemia and glomerular hyperfiltration.     

Inhibition of in vitro angiogenesis by platelet factor-4-derived peptides and mechanism of action.

In this study, we examined in detail the interaction of platelet factor-4 (PF-4) with fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor (VEGF) and the effect of PF-4-derived synthetic peptides. We show that a peptide between amino acids 47 and 70 that contains the heparin-binding lysine-rich site inhibits FGF-2 or VEGF function. This is based on the following observations: PF-4 peptide 47-70 inhibited FGF-2 or VEGF binding to endothelial cells; it inhibited FGF-2 or VEGF binding to FGFRs or VEGFRs in heparan sulfate-deficient CHO cells transfected with FGFR1 (CHOFGFR1) or VEGFR2 (CHOmVEGFR2) cDNA; it blocked proliferation or tube formation in three-dimensional angiogenesis assays; and, finally, it competed with the direct association of (125)I-PF-4 with FGF-2 or VEGF, respectively, and inhibited heparin-induced FGF-2 dimerization. A shorter C-terminal peptide (peptide 58-70), which still contained the heparin-binding lysin-rich site, had no effect. Peptide 17-58, which is located in the central part of the molecule, although it does not inhibit FGF-2 or VEGF binding or biologic activity in endothelial cells, inhibited heparin-dependent binding of (125)I-FGF-2 or (125)I-VEGF to CHOmFGFR1 or CHOmVEGFR2 cells, respectively. Shorter peptides (peptides 34-58 and 47-58) did not show any of these effects.     

Thrombospondin-2: a potent endogenous inhibitor of tumor growth and angiogenesis

Recent evidence suggests a potential role for thrombospondin-2 (TSP-2), a matricellular glycoprotein, in the regulation of primary angiogenesis. To directly examine the biological effect of TSP-2 expression on tumor growth and angiogenesis, human A431 squamous cell carcinoma cells, which do not express TSP-2, were stably transfected with a murine TSP-2 expression vector or with vector alone. A431 cells expressing TSP-2 did not show an altered growth rate, colony-forming ability, or susceptibility to induction of apoptosis in vitro. However, injection of TSP-2-transfected clones into the dermis of nude mice resulted in pronounced inhibition of tumor growth that was significantly stronger than the inhibition observed in A431 clones stably transfected with a thrombospondin-1 (TSP-1) expression vector, and combined overexpression of TSP-1 and TSP-2 completely prevented tumor formation. Extensive areas of necrosis were observed in TSP-2-expressing tumors, and both the density and the size of tumor vessels were significantly reduced, although tumor cell expression of the major tumor angiogenesis factor, vascular endothelial growth factor, was maintained at high levels. These findings establish TSP-2 as a potent endogenous inhibitor of tumor growth and angiogenesis.     

Role of chemokines in angiogenesis: CXCL12/SDF-1 and CXCR4 interaction,a key regulator of endothelial cell responses

Chemokines are small proteins that act as cell attractants via the activation of G protein-coupled receptors. Chemokines play an important role in several pathophysiological processes such as inflammation and immunity. Many proinflammatory chemokines also support the development of vascular blood supply at the site of inflammation. Similarly, tumor-generated chemokines can contribute to tumor growth by promoting angiogenesis. Recently, significant advances have been made in understanding the contribution of chemokines to the angiogenesis process. This review will discuss first the evidence supporting the direct contribution of different chemokine subfamily members, including CC, CXC, and CX3C chemokines, as positive or negative regulators of the angiogenesis process based on the expression of their cognate receptors on endothelial cells. Additionally, the relationship between classic angiogenic factors and chemokine receptor expression on endothelial cells, and the implications of chemokine production by cancer cells will be analyzed with particular emphasis on the CXCL12/stromal-cell derived factor-1 interaction with CXCR4.     

Effects of cytochalasin H, a potent inhibitor of cytoskeletal reorganisation, on platelet function

Platelets contain a well-developed and dynamic cytoskeleton composed mainly of actin and actin-associated proteins. Upon platelet activation there is rapid polymerisation of actin and a marked reorganisation of the platelet cytoskeleton. Cytochalasins are agents that interfere with the polymerisation of actin, and it has recently been discovered that cytochalasin H (CyH) is particularly effective as an inhibitor of the cytoskeletal reorganisation that occurs in platelets following activation by adenosine diphosphate (ADP). Here we have used CyH to inhibit platelet cytoskeletal reorganisation and to determine its effects on various aspects of platelet function. Experiments were performed in hirudinized platelet-rich plasma (PRP) or whole blood obtained from human volunteers. PRP was treated with 10 microM CyH or vehicle, then activated by ADP. The effect of CyH on cytoskeletal reorganisation was determined by SDS-PAGE of the Triton X-100 insoluble cytoskeletons and quantitated by densitometry. Platelet aggregation and aggregate stability in PRP were measured by monitoring changes in light absorbance; aggregation was measured in whole blood via platelet counting. Shape change, P-selectin expression and changes in intracellular calcium were measured using flow cytometry. CyH prevented the normal incorporation of actin, alpha-actinin and actin-binding protein into the cytoskeleton that occurred following ADP activation, and incorporation of myosin was markedly reduced. Aggregation was only partially inhibited but, more dramatically, the rate of disaggregation following addition of certain agents that interfere with fibrinogen binding to glycoprotein IIb/IIIa on the surface of platelets was markedly increased. The ADP-induced shape change was also inhibited. CyH had no effect on calcium mobilisation. Curiously, expression of P-selectin was potentiated by CyH, suggesting a modulatory role of the cytoskeleton in platelet secretory activity. The results suggest that cytoskeletal reorganisation plays an important role in platelet shape change and aggregation and contributes in a major way to the stability of the aggregates that form.