Intercellular transfer regulation of the paracrine activity of GPI-anchored Cripto-1 as a Nodal co-receptor

Cripto-1 (CR-1) is a glycosylphosphatidylinositol-anchored glycoprotein which acts as an obligate co-receptor of a TGFβ family ligand, Nodal. Previous studies have demonstrated that CR-1 functions in a paracrine fashion by a cellular mechanism which has not been fully described. This paracrine activity was observed only when CR-1 was expressed as a membrane-bound form and was abolished when CR-1 was expressed in a soluble form. In the current study, we found that there were few biochemical differences in post-translational modifications between membrane-anchored and soluble forms of CR-1. Flow cytometric analysis revealed an intercellular transfer of the membrane-bound form of CR-1 between cells. CR-1-expressing cells formed unique membrane extensions, generated more membrane fragments than control cells, and exhibited enhanced cellular adhesion. Thus, expression of CR-1 may alter the physiochemical properties of the plasma membrane resulting in an enhancement of intercellular transfer of cellular signaling components which may account for the paracrine activity of CR-1.     

Cleavage of carcinoembryonic antigen induces metastatic potential in colorectal carcinoma

Carcinoembryonic antigen (CEA), a widely used tumor marker, is attached by a glycosylphosphatidylinositol (GPI) anchor motif to the cell membrane. Recent study suggested that membrane-bound CEA might be cleaved by glycosylphosphatidylinositol-phospholipase D (GPI-PLD). We studied the effect of GPI-PLD on the cleavage of CEA to elucidate the implication for metastatic potential in colorectal carcinoma cells. CEA amount of conditioned medium was changed by suramin and phenanthroline (activator and inhibitor of GPI-PLD) only in SW620 and SW837 which expressed both CEA and GPI-PLD mRNA. Suramin treatment also augmented migratory activity and decreased cell surface CEA expression in SW620 and SW837. Furthermore, GPI-PLD knockdown cells using GPI-PLD-specific siRNA in SW620 and SW837 showed decreased CEA secretion from cell membrane and the migration activity, increased membrane-bound CEA amount. Splenic injection of SW620 and SW837 induced marked hepatic metastases in nude mice. These results suggest that membrane-bound CEA is cleaved by GPI-PLD and that this cleavage enhances the metastatic potential in colorectal carcinoma cells.     

Cellular glycosylphosphatidylinositol-specific phospholipase D regulates urokinase receptor shedding and cell surface expression

The glycosylphosphatidylinositol (GPI) - anchored, multifunctional receptor for the serine proteinase, urokinase plasminogen activator (uPAR, CD87), regulates plasminogen activation and cell migration, adhesion, and proliferation. uPAR occurs in functionally distinct, membrane-anchored and soluble isoforms (s-uPAR) in vitro and in vivo. Recent evidence indicates that s-uPAR present in the circulation of cancer patients correlates with tumor malignancy and represents a valuable prognostic marker in certain types of cancer. We have therefore analyzed the mechanism of uPAR shedding in vitro. We present evidence that uPAR is actively released from ovarian cancer cells since the rate of receptor shedding did not correlate with uPAR expression. While s-uPAR was derived from the cell surface, it lacked the hydrophobic portion of the GPI moiety indicating anchor cleavage. We show that uPAR release is catalyzed by cellular GPI-specific phospholipase D (GPI-PLD), an enzyme cleaving the GPI anchor of the receptor. Thus, recombinant GPI-PLD expression increased receptor release up to fourfold. Conversely, a 40% reduction in GPI-PLD activity by GPI-PLD antisense mRNA expression inhibited uPAR release by more than 60%. We found that GPI-PLD also regulated uPAR expression, possibly by releasing a GPI-anchored growth factor. Our data suggest that cellular GPI-PLD might be involved in the generation of circulating prognostic markers in cancer and possibly regulate the function of GPI-anchored proteins by generating functionally distinct, soluble counterparts. J. Cell. Physiol. 180:225–235, 1999. © 1999 Wiley-Liss, Inc.     

Novel Processed Form of Syndecan-1 Shed from SCC-9 Cells Plays a Role in Cell Migration

The extracellular milieu is comprised in part by products of cellular secretion and cell surface shedding. The presence of such molecules of the sheddome and secretome in the context of the extracellular milieu may have important clinical implications. In cancer they have been hypothesized to play a role in tumor growth and metastasis. The objective of this study was to evaluate whether the sheddome/secretome from two cell lines could be correlated with their potential for tumor development. Two epithelial cell lines, HaCaT and SCC-9, were chosen based on their differing abilities to form tumors in animal models of tumorigenesis. These cell lines when stimulated with phorbol-ester (PMA) showed different characteristics as assessed by cell migration, adhesion and higher gelatinase activity. Proteomic analysis of the media from these treated cells identified interesting, functionally relevant differences in their sheddome/secretome. Among the shed proteins, soluble syndecan-1 was found only in media from stimulated tumorigenic cells (SCC-9) and its fragments were observed in higher amount in the stimulated tumorigenic cells than stimulated non-tumorigenic cells (HaCaT). The increase in soluble syndecan-1 was associated with a decrease in membrane-bound syndecan-1 of SCC-9 cells after PMA stimuli. To support a functional role for soluble syndecan-1 fragments we demonstrated that the synthetic syndecan-1 peptide was able to induce cell migration in both cell lines. Taken together, these results suggested that PMA stimulation alters the sheddome/secretome of the tumorigenic cell line SCC-9 and one such component, the syndecan-1 peptide identified in this study, was revealed to promote migration in these epithelial cell lines.     

Fractalkine is expressed by smooth muscle cells in response to IFN-gamma and TNF-alpha and is modulated by metalloproteinase activity.

Fractalkine/CX3C-chemokine ligand 1 is expressed as a membrane-spanning adhesion molecule that can be cleaved from the cell surface to produce a soluble chemoattractant. Within the vasculature, fractalkine is known to be generated by endothelial cells, but to date there are no reports describing its expression by smooth muscle cells (SMC). In this study we demonstrate that IFN-gamma and TNF-alpha, but not IL-1beta, cooperate synergistically to induce fractalkine mRNA and protein expression in cultured aortic SMC. We also report the release of functional, soluble fractalkine from the membranes of stimulated SMC. This release is inhibited by the zinc metalloproteinase inhibitor batimastat, resulting in the accumulation of membrane-associated fractalkine on the SMC surface. Therefore, an SMC-derived metalloproteinase activity is involved in fractalkine shedding. While soluble fractalkine present in SMC-conditioned medium is capable of inducing calcium transients in cells expressing the fractalkine receptor (CX3CR1), blocking experiments using neutralizing Abs reveal that it can be inactivated without affecting the chemotactic activity of SMC-conditioned media on monocytes. However, membrane-bound fractalkine plays a major role in promoting adhesion of monocytic cells to activated SMC. This fractalkine-mediated adhesion is further enhanced in the presence of batimastat, indicating that shedding of fractalkine from the cell surface down-regulates the adhesive properties of SMC. Hence, during vascular inflammation, the synergistic induction of fractalkine by IFN-gamma and TNF-alpha together with its metalloproteinase-mediated cleavage may finely control the recruitment of monocytes to SMC within the blood vessel wall.     

Plasminogen activator inhibitor-1 supports IL-8-mediated neutrophil transendothelial migration by inhibition of the constitutive shedding of endothelial IL-8/heparan sulfate/syndecan-1 complexes

The endothelium is the primary barrier to leukocyte recruitment at sites of inflammation. Neutrophil recruitment is directed by transendothelial gradients of IL-8 that, in vivo, are bound to the endothelial cell surface. We have investigated the identity and function of the binding site(s) in an in vitro model of neutrophil transendothelial migration. In endothelial culture supernatants, IL-8 was detected in a trimolecular complex with heparan sulfate and syndecan-1. Constitutive shedding of IL-8 in this form was increased in the presence of a neutralizing Ab to plasminogen activator inhibitor-1 (PAI-1), indicating a role for endothelial plasminogen activator in the shedding of IL-8. Increased shedding of IL-8/heparan sulfate/syndecan-1 complexes was accompanied by inhibition of neutrophil transendothelial migration, and aprotinin, a potent plasmin inhibitor, reversed this inhibition. Platelets, added as an exogenous source of PAI-1, had no effect on shedding of the complexes or neutrophil migration. Our results indicate that IL-8 is immobilized on the endothelial cell surface through binding to syndecan-1 ectodomains, and that plasmin, generated by endothelial plasminogen activator, induces the shedding of this form of IL-8. PAI-1 appears to stabilize the chemoattractant form of IL-8 at the cell surface and may represent a therapeutic target for novel anti-inflammatory strategies.     

Stimulated shedding of vascular cell adhesion molecule 1 (VCAM-1) is mediated by tumor necrosis factor-alpha-converting enzyme (ADAM 17)

A variety of cell surface adhesion molecules can exist as both transmembrane proteins and soluble circulating forms. Increases in the levels of soluble adhesion molecules have been correlated with a variety of inflammatory diseases, suggesting a pathological role. Although soluble forms are thought to result from proteolytic cleavage from the cell surface, relatively little is known about the proteases responsible for their release. In this report we demonstrate that under normal culture conditions, cells expressing vascular cell adhesion molecule 1 (VCAM-1) release a soluble form of the extracellular domain that is generated by metalloproteinase-mediated cleavage. VCAM-1 release can be rapidly simulated by phorbol 12-myristate 13-acetate (PMA), and this induced VCAM-1 shedding is mediated by metalloproteinase cleavage of VCAM-1 near the transmembrane domain. PMA-induced VCAM-1 shedding occurs as the result of activation of a specific pathway, as the generation of soluble forms of three other adhesion molecules, E-selectin, platelet-endothelial cell adhesion molecule 1, and intercellular adhesion molecule 1, are not altered by PMA stimulation. Using cells derived from genetically deficient mice, we identify tumor necrosis factor-alpha-converting enzyme (TACE or ADAM 17) as the protease responsible for PMA-induced VCAM-1 release, including shedding of endogenously expressed VCAM-1 by murine endothelial cells. Therefore, TACE-mediated shedding of VCAM-1 may be important for the regulation of VCAM-1 function at the cell surface.     

Enzymatic Release of Zn2+-Glycerophosphocholine Cholinephosphodiesterase from Brain Membranes by Glycosylphosphatidylinositol-Specific Phospholipases and its Regulation

Enzymatic conversion of glycosylphosphatidylinositol (GPI)-linked Zn²⁺-glycerophosphocholine phosphodiesterase was investigated. The activity of glycosylphosphatidylinositol-specific phospholipase-D (GPI-PLD), based on the conversion of amphiphilic form of phosphodiesterase into hydrophilic form, showing an optimum pH of about pH 6.6, increased continuously until 60 min. The activity of membrane-bound GPI-PL, based on the formation of hydrophilic form of phosphodiesterase, exhibiting an optimum pH of 7.4, increased up to 30 min, and reached a plateau. Inhibition studies indicate that while GPI-PLD activity was generally sensitive to ionic bio-detergents, it was not inhibited by myristoyl glycerol, a neutal detergent. Meanwhile, the membrane-bound GPI-PL was not affected remarkably by these detergents except that myristoyl glycerol expressed a modest increase of activity of membrane bound GPI-PL. In addition, the membrane-bound GPI-PL appeared to be enhanced by by suramin or oleic acid, which strongly inhibited GPI-PLD. From this results, it is suggested that in brain there may be two phospholipases responsible for the conversion of membrane-bound GPI-anchors to hydrophilic forms, and that this conversion might be regulated by endogenous lipids.     

Knockdown of Cripto-1 inhibits the proliferation,migration, invasion,and angiogenesis in prostate carcinoma cells

Cripto-1 (CR-1) is a member of the epidermal growth factor-Cripto-1/FRL1/Cryptic gene family that plays a key role in the various malignant cancers. However, the role of CR-1 in prostate carcinoma (PCa) remains limited. The expression of CR-1 was down-regulated by small interfering RNA (siRNA). Western blot measured the expression levels of CR-1 and some related proteins. We performed Cell Counting Kit-8, 5-ethynyl-2-deoxyuridine (EdU) incorporation assay and flow cytometry to detect the cellular proliferation and cycle. The transwell assay was used to observe cellular migration and invasion. The ability of angiogenesis was evaluated by tube formation assay. Our results showed that CR-1 knockdown markedly inhibited cell proliferation and induced cycle arrest in G1 phase, as p21 and p27 were up-regulated, whereas cyclin D1 and cyclin E1 were diminished. Moreover, silencing of CR-1 dramatically inhibited cell migration and invasion, repressed matrix metalloproteinases, and disturbed epithelial-mesenchymal transition. CR-1 siRNA suppressed the secreted level of vascular endothelial growth factor, and reduced protein level of Vascular endothelial growth factor receptor 2. We further found that decreased CR-1 expression inhibited FAK/Src/PI3K and Wnt/β-catenin signalling in PCa cells. These results suggested CR-1 might be served as an effective therapeutic target in PCa.     

Sodium arsenite down-regulates the expression of X-linked inhibitor of apoptosis protein via translational and post-translational mechanisms in hepatocellular carcinoma

Amphiregulin (AREG), an EGF family protein, is synthesized as a type I transmembrane precursor (proAREG) and expressed on the cell surface with an extracellular EGF-like domain and an intracellular short cytoplasmic tail. The ectodomain shedding yields a soluble EGF receptor ligand (soluble AREG) which binds to EGF receptor (EGFR) and concomitantly induces migration of unshed proAREG from the plasma membrane to the nuclear envelope (NE). AREG is known to play a potential role in breast cancer and has been intensively investigated as an EGF receptor ligand, while the function of the NE-localized proAREG remains unknown. In this study we used a truncated mutant that mimics NE-localized proAREG without shedding stimuli to discriminate between the functions of NE-localized and plasma membrane-localized proAREG and demonstrate that NE-localized proAREG activates breast cancer cell migration, but suppresses cell growth. Moreover, the present study shows that induction of cell migration by NE-localized proAREG does not require the extracellular growth factor domain or EGF receptor function. Collectively these data demonstrate a novel function mediated by the intracellular domain of proAREG and suggest a significant role for NE-localized proAREG in driving human breast cancer progression.     

Release of GPI-anchored membrane proteins by a cell-associated GPI-specific phospholipase D.

Although many glycosylphosphatidylinositol (GPI)-anchored proteins have been observed as soluble forms, the mechanisms by which they are released from the cell surface have not been demonstrated. We show here that a cell-associated GPI-specific phospholipase D (GPI-PLD) releases the GPI-anchored, complement regulatory protein decay-accelerating factor (DAF) from HeLa cells, as well as the basic fibroblast growth factor-binding heparan sulfate proteoglycan from bone marrow stromal cells. DAF found in the HeLa cell culture supernatants contained both [3H]ethanolamine and [3H]inositol, but not [3H]palmitic acid, whereas the soluble heparan sulfate proteoglycan present in bone marrow stromal cell culture supernatants contained [3H]ethanolamine. 125I-labeled GPI-DAF incorporated into the plasma membranes of these two cell types was released in a soluble form lacking the fatty acid GPI-anchor component. GPI-PLD activity was detected in lysates of both HeLa and bone marrow stromal cells. Treatment of HeLa cells with 1,10-phenanthroline, an inhibitor of GPI-PLD, reduced the release of [3H]ethanolamine-DAF by 70%. The hydrolysis of these GPI-anchored molecules is likely to be mediated by an endogenous GPI-PLD because [3H]ethanolamine DAF is constitutively released from HeLa cells maintained in serum-free medium. Furthermore, using PCR, a GPI-PLD mRNA has been identified in cDNA libraries prepared from both cell types. These studies are the first demonstration of the physiologically relevant release of GPI-anchored proteins from cells by a GPI-PLD.     

Regulated shedding of transmembrane chemokines by the disintegrin and metalloproteinase 10 facilitates detachment of adherent leukocytes

CX3CL1 (fractalkine) and CXCL16 are unique members of the chemokine family because they occur not only as soluble, but also as membrane-bound molecules. Expressed as type I transmembrane proteins, the ectodomain of both chemokines can be proteolytically cleaved from the cell surface, a process known as shedding. Our previous studies showed that the disintegrin and metalloproteinase 10 (ADAM10) mediates the largest proportion of constitutive CX3CL1 and CXCL16 shedding, but is not involved in the phorbolester-induced release of the soluble chemokines (inducible shedding). In this study, we introduce the calcium-ionophore ionomycin as a novel, very rapid, and efficient inducer of CX3CL1 and CXCL16 shedding. By transfection in COS-7 cells and ADAM10-deficient murine embryonic fibroblasts combined with the use of selective metalloproteinase inhibitors, we demonstrate that the inducible generation of soluble forms of these chemokines is dependent on ADAM10 activity. Analysis of the C-terminal cleavage fragments remaining in the cell membrane reveals multiple cleavage sites used by ADAM10, one of which is preferentially used upon stimulation with ionomycin. In adhesion studies with CX3CL1-expressing ECV-304 cells and cytokine-stimulated endothelial cells, we demonstrate that induced CX3CL1 shedding leads to the release of bound monocytic cell lines and PBMC from their cellular substrate. These data provide evidence for an inducible release mechanism via ADAM10 potentially important for leukocyte diapedesis.     

Membrane-bound ICAM-1 is upregulated by trypsin and contributes to leukocyte migration in acute pancreatitis

In acute pancreatitis, ICAM-1 is upregulated in various organs and contributes to the development of organ injury. To investigate the effects of pancreatic proteases on ICAM-1 expression and their role in the early process of leukocyte migration, human umbilical vein endothelial cells (HUVECs) were incubated with serum subjected to limited trypsin digestion and Wistar rats were injected with trypsin. Significant upregulation of membrane-bound ICAM-1 was seen on HUVECs incubated with trypsinated serum. Likewise, soluble ICAM-1 increased in the supernatant of HUVECs. Changes of membrane-bound ICAM-1 and soluble ICAM-1 were maximal with high concentrations of trypsin. HUVECs incubated with TNF-alpha (positive control) showed similar changes. In the pancreas and lungs of animals infused with trypsin, ICAM-1 and leukocyte sequestration were increased compared with controls. Reflecting the relevance of protease-induced ICAM-1 expression in leukocyte migration, leukocyte-endothelium interaction, as assessed by intravital microscopy, was markedly increased by trypsin. Inhibition of ICAM-1 ameliorated these changes significantly. In conclusion, trypsinated serum induces the upregulation of both membrane-bound ICAM-1 on endothelial cells and soluble ICAM-1. These changes contribute to the early steps of leukocyte migration in acute pancreatitis. The role of soluble ICAM-1 remains to be investigated.     

Membrane Cholesterol Modulates LOX-1 Shedding in Endothelial Cells

The lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a scavenger receptor responsible for ox-LDL recognition, binding and internalization, which is up-regulated during atherogenesis. Its activation triggers endothelium dysfunction and induces inflammation. A soluble form of LOX-1 has been identified in the human blood and its presence considered a biomarker of cardiovascular diseases. We recently showed that cholesterol-lowering drugs inhibit ox-LDL binding and internalization, rescuing the ox-LDL induced apoptotic phenotype in primary endothelial cells. Here we have investigated the molecular bases of human LOX-1 shedding by metalloproteinases and the role of cell membrane cholesterol on the regulation of this event by modulating its level with MβCD and statins. We report that membrane cholesterol affects the release of different forms of LOX-1 in cells transiently and stably expressing human LOX-1 and in a human endothelial cell line (EA.hy926). In particular, our data show that i) cholesterol depletion triggers the release of LOX-1 in exosomes as a full-length transmembrane isoform and as a truncated ectodomain soluble fragment (sLOX-1); ii) endothelial cells secrete a soluble metalloproteinase which induces LOX-1 ectodomain shedding and iii) long term statins treatment enhances sLOX-1 proteolytic shedding.     

Membrane type 1-matrix metalloproteinase is activated during migration of human endothelial cells and modulates endothelial motility and matrix remodeling.

Matrix metalloproteinases are thought to play an important role in endothelial cell migration and matrix remodeling. We have used an in vitro wound healing migration model and newly generated anti-membrane type 1-matrix metalloproteinase (MT1-MMP) monoclonal antibodies (mAbs) to characterize the role of MT1-MMP during this process. First, the expression and shedding of MT1-MMP are up-regulated upon induction of migration in endothelial cells, as demonstrated by flow cytometry and Western blot analysis. Furthermore, MT1-MMP is concentrated at discrete areas in migrating endothelial cells, in contrast to the diffuse pattern observed in confluent cells. Interestingly, migration of endothelial cells results in the stimulation of MT1-MMP activity, as shown by its ability to process pro-MMP-2 and to degrade fibrinogen assessed by zymography. Moreover, MT1-MMP-mediated gelatin degradation is enriched at migration sites. mAbs generated against the MT1-MMP catalytic domain are shown to inhibit MT1-MMP enzymatic activity and to impair both phorbol 12-myristate 13-acetate-induced endothelial migration and invasion of collagen and fibrin gels. Furthermore, a reduction in the formation of capillary tubes in Matrigel is also observed when endothelial cells are pretreated with the blocking anti-MT1-MMP mAbs. Altogether, these data demonstrate that MT1-MMP plays an important role during endothelial cell migration, and its activity can modulate endothelial migration, invasion, and formation of capillary tubes during the angiogenic response.     

CD154 Is Released from T-cells by a Disintegrin and Metalloproteinase Domain-containing Protein 10 (ADAM10) and ADAM17 in a CD40 Protein-dependent Manner

CD154 (CD40 ligand) is a type II transmembrane protein that belongs to the tumor necrosis factor superfamily. The soluble form of CD154 (sCD154), which results from the shedding of membrane-bound CD154, plays a key role in the production of proinflammatory cytokines and has been linked to various autoimmune and vascular disorders. Therefore, elucidating the mechanisms by which CD154 is released from the cell surface following its interaction with its various receptors is of primordial importance. Using co-culture experiments, we show that CD154 is shed predominantly upon its engagement with CD40. Indeed, only CD40 (both membrane-bound and soluble) and not α5β1 or αMβ2 is involved in the cleavage and release of CD154 from Jurkat E6.1 T-cells. Interestingly, CD154 is cleaved independently of the formation of cell surface CD40 homodimers and independently of its association into lipid rafts. In contrast, we found that the protein kinase C (PKC) signaling family and the matrix metalloproteinases ADAM10 and ADAM17 are intimately involved in this process. In conclusion, our data indicate that CD154 is released from T-cells by ADAM10 and ADAM17 upon CD40 ligation. These findings add significant insights into the mechanisms by which CD154 is down-regulated and may lead to the generation of novel therapeutic targets for the treatment of CD154-associated disorders.     

Negative regulation of osteoclastogenesis by ectodomain shedding of receptor activator of NF-kappaB ligand

Receptor activator of NF-kappaB ligand (RANKL) is a transmembrane glycoprotein that has an essential role in the development of osteoclasts. The extracellular portion of RANKL is cleaved proteolytically to produce soluble RANKL, but definite RANKL sheddase(s) and the physiologic function of RANKL shedding have not yet been determined. In the present study, we found that matrix metalloproteinase (MMP) 14 and a disintegrin and metalloproteinase (ADAM) 10 have strong RANKL shedding activity. In Western blot analysis, soluble RANKL was detected as two different molecular weight products, and RNA interference of MMP14 and ADAM10 resulted in a reduction of both the lower and higher molecular weight products. Suppression of MMP14 in primary osteoblasts increased membrane-bound RANKL and promoted osteoclastogenesis in cocultures with macrophages. Soluble RANKL produced by osteoblasts from MMP14-deficient mice was markedly reduced, and their osteoclastogenic activity was promoted, consistent with the findings of increased osteoclastogenesis in vivo. RANKL shedding is an important process that down-regulates local osteoclastogenesis.     

A truncated recombinant intercellular adhesion molecule-1 inhibits adhesion of leukemic cell lines to upregulated endothelial cells

Summary Intercellular adhesion molecule-1, a member of the immunoglobulin supergene family, is the ligand for the integrin lymphocyte function associated antigen-1. Intercellular adhesion molecule-1 and lymphocyte function associated antigen-1 binding interactions mediate leukocyte adherence and migration. Previous work has shown that the adherence of lymphocyte function associated antigen-1 is directed to the first immunoglobulinlike domain of the endothelial cell surface protein intracellular adhesion molecule-1. We have constructed a truncated intercellular adhesion molecule-1 gene encoding the first 185 amino acids from the amino terminus and overexpressed it inEscherichia coli. The recombinant protein was purified from insoluble inclusion bodies and refolded into an active conformation by a denaturation/renaturation cycle. The identity of the protein was confirmed by microsequencing and by Western blot analysis using a polyclonal antibody to ICAM-1. We have demonstrated that this soluble region of the otherwise membrane-bound ligand is an inhibitor of Molt or HL-60 cell adhesion to cytokine-stimulated endothelial cells.     

Angiopoietin-2 stimulates migration of endothelial progenitors and their interaction with endothelium

The factors controlling recruitment of endogenous and transplanted endothelial progenitor cells (EPC) to areas of neovascularization are largely unknown. In this study, we have examined the possibility that EPC migration and adhesion could be regulated by angiopoietin-2 (Ang2), a soluble ligand expressed by endothelial cells at sites of vessel remodelling and angiogenesis. We show for the first time that Ang2 causes a marked stimulation of EPC migration. This was specific for EPC as the ligand failed to affect endothelial cell migration. Ang2-stimulated EPC migration was inhibited by soluble Tie2 ectodomain. Furthermore, the ligand stimulated adhesion between EPC and endothelial monolayers.