Anna Janowska-Wieczorek

Membrane-derived microvesicles: important and underappreciated mediators of cell-to-cell communication.

Normal and malignant cells shed from their surface membranes as well as secrete from the endosomal membrane compartment circular membrane fragments called microvesicles (MV). MV that are released from viable cells are usually smaller in size compared to the apoptotic bodies derived from damaged cells and unlike them do not contain fragmented DNA. Growing experimental evidence indicates that MV are an underappreciated component of the cell environment and play an important pleiotropic role in many biological processes. Generally, MV are enriched in various bioactive molecules and may (i) directly stimulate cells as a kind of ‘signaling complex’, (ii) transfer membrane receptors, proteins, mRNA and organelles (e.g., mitochondria) between cells and finally (iii) deliver infectious agents into cells (e.g., human immuno deficiency virus, prions). In this review, we discuss the pleiotropic effects of MV that are important for communication between cells, as well as the role of MV in carcinogenesis, coagulation, immune responses and modulation of susceptibility/infectability of cells to retroviruses or prions.

Trafficking of normal stem cells and metastasis of cancer stem cells involve similar mechanisms: pivotal role of the SDF-1-CXCR4 axis.

The α‐chemokine stromal‐derived factor (SDF)‐1 and the G‐protein–coupled seven‐span transmembrane receptor CXCR4 axis regulates the trafficking of various cell types. In this review, we present the concept that the SDF‐1–CXCR4 axis is a master regulator of trafficking of both normal and cancer stem cells. Supporting this is growing evidence that SDF‐1 plays a pivotal role in the regulation of trafficking of normal hematopoietic stem cells (HSCs) and their homing/retention in bone marrow. Moreover, functional CXCR4 is also expressed on nonhematopoietic tissue‐committed stem/progenitor cells (TCSCs); hence, the SDF‐1–CXCR4 axis emerges as a pivotal regulator of trafficking of various types of stem cells in the body. Furthermore, because most if not all malignancies originate in the stem/progenitor cell compartment, cancer stem cells also express CXCR4 on their surface and, as a result, the SDF‐1–CXCR4 axis is also involved in directing their trafficking/metastasis to organs that highly express SDF‐1 (e.g., lymph nodes, lungs, liver, and bones). Hence, we postulate that the metastasis of cancer stem cells and trafficking of normal stem cells involve similar mechanisms, and we discuss here the common molecular mechanisms involved in these processes. Finally, the responsiveness of CXCR4+ normal and malignant stem cells to an SDF‐1 gradient may be regulated positively/primed by several small molecules related to inflammation which enhance incorporation of CXCR4 into membrane lipid rafts, or may be inhibited/blocked by small CXCR4 antagonist peptides. Consequently, strategies aimed at modulating the SDF‐1–CXCR4 axis could have important clinical applications both in regenerative medicine to deliver normal stem cells to the tissues/organs and in clinical hematology/oncology to inhibit metastasis of cancer stem cells.

Migration of Bone Marrow and Cord Blood Mesenchymal Stem Cells In Vitro Is Regulated by Stromal‐Derived Factor‐1‐CXCR4 and Hepatocyte Growth Factor‐c‐met Axes and Involves Matrix Metalloproteinases

Human mesenchymal stem cells (MSCs) are increasingly being considered in cell‐based therapeutic strategies for regeneration of various organs/tissues. However, the signals required for their homing and recruitment to injured sites are not yet fully understood. Because stromal‐derived factor (SDF)‐1 and hepatocyte growth factor (HGF) become up‐regulated during tissue/organ damage, in this study we examined whether these factors chemoattract ex vivo‐expanded MSCs derived from bone marrow (BM) and umbilical cord blood (CB). Specifically, we investigated the expression by MSCs of CXCR4 and c‐met, the cognate receptors of SDF‐1 and HGF, and their functionality after early and late passages of MSCs. We also determined whether MSCs express matrix metalloproteinases (MMPs), including membrane type 1 (MT1)‐MMP, matrix‐degrading enzymes that facilitate the trafficking of hematopoietic stem cells. We maintained expanded BM‐ or CB‐derived MSCs for up to 15–18 passages with monitoring of the expression of 1) various tissue markers (cardiac and skeletal muscle, neural, liver, and endothelial cells), 2) functional CXCR4 and c‐met, and 3) MMPs. We found that for up to 15–18 passages, both BM‐ and CB‐derived MSCs 1) express mRNA for cardiac, muscle, neural, and liver markers, as well as the vascular endothelial (VE) marker VE‐cadherin; 2) express CXCR4 and c‐met receptors and are strongly attracted by SDF‐1 and HGF gradients; 3) express MMP‐2 and MT1‐MMP transcripts and proteins; and 4) are chemo‐invasive across the reconstituted basement membrane Matrigel. These in vitro results suggest that the SDF‐1‐CXCR4 and HGF‐c‐met axes, along with MMPs, may be involved in recruitment of expanded MSCs to damaged tissues.

Microvesicles derived from activated platelets induce metastasis and angiogenesis in lung cancer.

The role of platelets in tumor progression and metastasis has been recognized but the mechanism of their action remains unclear. Five human lung cancer cell lines (A549, CRL 2066, CRL 2062, HTB 183, HTB 177) and a murine Lewis lung carcinoma (LCC) cell line (for an in vivo model of metastasis) were used to investigate how platelet‐derived microvesicles (PMV), which are circular fragments shed from the surface membranes of activated platelets, and exosomes released from platelet α‐granules, could contribute to metastatic spread. We found that PMV transferred the platelet‐derived integrin CD41 to most of the lung cancer cell lines tested and stimulated the phosphorylation of mitogen‐activated protein kinase p42/44 and serine/threonine kinase as well as the expression of membrane type 1‐matrix metalloproteinase (MT1‐MMP). PMV chemoattracted 4 of the 5 cell lines, with the highly metastatic A549 cells exhibiting the strongest response. In A549 cells, PMV were shown to stimulate proliferation, upregulate cyclin D2 expression and increase trans‐Matrigel chemoinvasion. Furthermore, in these cells, PMV stimulated mRNA expression for angiogenic factors such as MMP‐9, vascular endothelial growth factor, interleukin‐8 and hepatocyte growth factor, as well as adhesion to fibrinogen and human umbilical vein endothelial cells. Intravenous injection of murine PMV‐covered LLC cells into syngeneic mice resulted in significantly more metastatic foci in their lungs and LLC cells in bone marrow than in control animals injected with LCC cells not covered with PMV. Based on these findings, we suggest that PMV play an important role in tumor progression/metastasis and angiogenesis in lung cancer.

Stem cell plasticity revisited: CXCR4-positive cells expressing mRNA for early muscle, liver and neural cells ‘hide out’ in the bone marrow

It has been suggested that bone marrow (BM)-derived hematopoietic stem cells transdifferentiate into tissue-specific stem cells (the so-called phenomenon of stem cell plasticity), but the possibility of committed tissue-specific stem cells pre-existing in BM has not been given sufficient consideration. We hypothesized that (i) tissue-committed stem cells circulate at a low level in the peripheral blood (PB) under normal steady-state conditions, maintaining a pool of stem cells in peripheral tissues, and their levels increase in PB during stress/tissue injury, and (ii) they could be chemoattracted to the BM where they find a supportive environment and that the SDF-1–CXCR4 axis plays a prominent role in the homing/retention of these cells to BM niches. We performed all experiments using freshly isolated cells to exclude the potential for ‘transdifferentiation’ of hematopoietic stem or mesenchymal cells associated with in vitro culture systems. We detected mRNA for various early markers for muscle (Myf-5, Myo-D), neural (GFAP, nestin) and liver (CK19, fetoprotein) cells in circulating (adherent cell-depleted) PB mononuclear cells (MNC) and increased levels of expression of these markers in PB after mobilization by G-CSF (as measured using real-time RT-PCR). Furthermore, SDF-1 chemotaxis combined with real-time RT-PCR analysis revealed that (i) these early tissue-specific cells reside in normal murine BM, (ii) express CXCR4 on their surface and (iii) can be enriched (up to 60 ×) after chemotaxis to an SDF-1 gradient. These cells were also highly enriched within purified populations of murine Sca-1+ BM MNC as well as of human CD34+-, AC133+- and CXCR4-positive cells. We also found that the expression of mRNA for SDF-1 is upregulated in damaged heart, kidney and liver. Hence our data provide a new perspective on BM not only as a home for hematopoietic stem cells but also a ‘hideout’ for already differentiated CXCR4-positive tissue-committed stem/progenitor cells that follow an SDF-1 gradient, could be mobilized into PB, and subsequently take part in organ/tissue regeneration.

Platelet-derived microparticles stimulate proliferation, survival, adhesion, and chemotaxis of hematopoietic cells.

OBJECTIVE Peripheral blood platelet-derived microparticles (PMPs) circulate in blood and may interact directly with target cells affecting their various biological functions. METHODS To investigate the effect of human PMPs on hematopoiesis, we first phenotyped them for expression of various surface molecules and subsequently studied various biological responses of normal stem/progenitor (CD34(+)) and more differentiated precursor cells as well as several leukemic cell lines to PMPs. RESULTS We found that, in addition to platelet-endothelium attachment receptors (CD41, CD61 and CD62), PMPs express G-protein-coupled seven transmembrane-span receptors such as CXCR4 and PAR-1; cytokine receptors including TNF-RI, TNF-RII, and CD95; and ligands such as CD40L and PF-4. Moreover, we found that several of these receptors could be transferred by PMPs to the membranes of normal as well as malignant cells and observed that PMPs: 1) chemoattract these cells, 2) increase their adhesion, proliferation, and survival, and 3) activate in these cells various intracellular signaling cascades including MAPK p42/44, PI-3K-AKT, and STAT proteins. The biological effects of PMPs were only partly reduced by heat inactivation or trypsin digest, indicating that, in addition to the protein components of PMPs, lipid components are also responsible for their biological activity. CONCLUSIONS We conclude that PMPs modulate biological functions of hematopoietic cells and postulate that they play an important but as yet not fully understood role in intercellular cross-talk in hematopoiesis. Further studies, however, are needed to identify the PMP components that exert specific biological effects.

Expression of Functional CXCR4 by Muscle Satellite Cells and Secretion of SDF-1 by Muscle-Derived Fibroblasts is Associated with the Presence of Both Muscle Progenitors in Bone Marrow and Hematopoietic Stem/Progenitor Cells in Muscles

We found that the murine cell lines C2C12 and G7 derived from muscle satellite cells, which are essential for muscle regeneration, express the functional CXCR4 receptor on their surface and that the specific ligand for this receptor, α‐chemokine stromal‐derived factor 1 (SDF‐1), is secreted in muscle tissue. These cell lines responded to SDF‐1 stimulation by chemotaxis, phosphorylation of mitogen‐activated protein kinase (MAPK) p42/44 and AKT serine‐threonine kinase, and calcium flux, confirming the functionality of the CXCR4 receptor. Moreover, supernatants derived from muscle fibroblasts chemoattracted both satellite cells and human CD34+ hematopoietic stem/progenitor cells. In a similar set of experiments, supernatants from bone marrow fibroblasts were found to chemoattract CXCR4+ satellite cells just as they chemoattract CD34+ cells. Moreover, preincubation of both muscle satellite cells and hematopoietic stem/progenitor CD34+ cells before chemotaxis with T140, a specific CXCR4 inhibitor, resulted in a significantly lower chemotaxis to media conditioned by either muscle‐ or bone marrow‐derived fibroblasts. Based on these observations, we postulate that the SDF‐1‐CXCR4 axis is involved in chemoattracting circulating CXCR4+ muscle stem/progenitor and circulating CXCR4+ hematopoietic CD34+ cells to both muscle and bone marrow tissues. Thus, it appears that tissue‐specific stem cells circulating in peripheral blood could compete for SDF‐1+ niches, and this would explain, without invoking the concept of stem cell plasticity, why hematopoietic colonies can be cultured from muscles and early muscle progenitors can be cultured from bone marrow.

The SDF‐1‐CXCR4 Axis Stimulates VEGF Secretion and Activates Integrins but does not Affect Proliferation and Survival in Lymphohematopoietic Cells

To better define the role HIV‐related chemokine receptor‐chemokine axes play in human hematopoiesis, we investigated the function of the CXCR4 and CCR5 receptors in human myeloid, T‐ and B‐lymphoid cell lines selected for the expression of these receptors (CXCR4+, CXCR4+ CCR5+, and CCR5+ cell lines). We evaluated the phosphorylation of MAPK p42/44, AKT, and STAT proteins and examined the ability of the ligands for these receptors (stromal‐derived factor‐1 [SDF‐1] and macrophage inflammatory protein‐1β [MIP‐1β]) to influence cell growth, apoptosis, adhesion, and production of vascular endothelial growth factors (VEGF), matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in these cell lines. We found that A) SDF‐1, after binding to CXCR4, activates multiple signaling pathways and that in comparison with the MIP‐1β‐CCR5 axis, plays a privileged role in hematopoiesis; B) SDF‐1 activation of the MAPK p42/44 pathway and the PI‐3K‐AKT axis does not affect proliferation and apoptosis but modulates integrin‐mediated adhesion to fibronectin, and C) SDF‐1 induces secretion of VEGF, but not of MMPs or TIMPs. Thus the role of SDF‐1 relates primarily to the interaction of lymphohematopoietic cells with their microenvironment and does not directly influence their proliferation or survival. We conclude that perturbation of the SDF‐1‐CXCR4 axis during HIV infection may affect interactions of hematopoietic cells with the hematopoietic microenvironment.

Elucidation of CXCR7-Mediated Signaling Events and Inhibition of CXCR4-Mediated Tumor Cell Transendothelial Migration by CXCR7 Ligands

CXCR7 binds chemokines CXCL11 (I-TAC) and CXCL12 (SDF-1) but does not act as a classical chemoattractant receptor. Using CCX771, a novel small molecule with high affinity and selectivity for CXCR7, we found that, although CXCR7 is dispensable for “bare filter” in vitro chemotaxis, CXCR7 plays an essential role in the CXCL12/CXCR4-mediated transendothelial migration (TEM) of CXCR4+CXCR7+ human tumor cells. Importantly, although CXCL11 is unable to stimulate directly the migration of these cells, it acts as a potent antagonist of their CXCL12-induced TEM. Furthermore, even though this TEM is driven by CXCR4, the CXCR7 ligand CCX771 is substantially more potent at inhibiting it than the CXCR4 antagonist AMD3100, which is more than 100 times weaker at inhibiting TEM when compared with its ability to block bare filter chemotaxis. Far from being a “silent” receptor, we show that CXCR7 displays early hallmark events associated with intracellular signaling. Upon cognate chemokine binding, CXCR7 associates with β-arrestin2, an interaction that can be blocked by CXCR7-specific mAbs. Remarkably, the synthetic CXCR7 ligand CCX771 also potently stimulates β-arrestin2 recruitment to CXCR7, with greater potency and efficacy than the endogenous chemokine ligands. These results indicate that CXCR7 can regulate CXCL12-mediated migratory cues, and thus may play a critical role in driving CXCR4+CXCR7+ tumor cell metastasis and tissue invasion. CXCR7 ligands, such as the chemokine CXCL11 and the newly described synthetic molecule CCX771, may represent novel therapeutic opportunities for the control of such cells.

Novel insight into stem cell mobilization-plasma sphingosine-1-phosphate is a major chemoattractant that directs the egress of hematopoietic stem progenitor cells from the bone marrow and its level in peripheral blood increases during mobilization due to activation of complement cascade/membrane attack complex.

The complement cascade (CC) becomes activated and its cleavage fragments play a crucial role in the mobilization of hematopoietic stem/progenitor cells (HSPCs). Here, we sought to determine which major chemoattractant present in peripheral blood (PB) is responsible for the egress of HSPCs from the bone marrow (BM). We noticed that normal and mobilized plasma strongly chemoattracts HSPCs in a stromal-derived factor-1 (SDF-1)-independent manner because (i) plasma SDF-1 level does not correlate with mobilization efficiency; (ii) the chemotactic plasma gradient is not affected in the presence of AMD3100 and (iii) it is resistant to denaturation by heat. Surprisingly, the observed loss of plasma chemotactic activity after charcoal stripping suggested the involvement of bioactive lipids and we focused on sphingosine-1-phosphate (S1P), a known chemoattracant of HSPCs. We found that S1P (i) creates in plasma a continuously present gradient for BM-residing HSPCs; (ii) is at physiologically relevant concentrations a chemoattractant several magnitudes stronger than SDF-1 and (iii) its plasma level increases during mobilization due to CC activation and interaction of the membrane attack complex (MAC) with erythrocytes that are a major reservoir of S1P. We conclude and propose a new paradigm that S1P is a crucial chemoattractant for BM-residing HSPCs and that CC through MAC induces the release of S1P from erythrocytes for optimal egress/mobilization of HSPCs.