Jacek Kijowski

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.

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

OBJECTIVEnPeripheral blood platelet-derived microparticles (PMPs) circulate in blood and may interact directly with target cells affecting their various biological functions.nnnMETHODSnTo 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.nnnRESULTSnWe 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.nnnCONCLUSIONSnWe 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.

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.

Biological significance of MAPK, AKT and JAK-STAT protein activation by various erythropoietic factors in normal human early erythroid cells

The aim of this study was to identify signal transduction pathways activated by erythropoietin (EpO) and erythropoietin co‐stimulatory factors (kit ligand), insulin‐like growth factor, thrombopoietin, interleukin 3 and granulocyte‐macrophage colony‐stimulating factor) in normal human bone marrow CD34+ cells and du200311 erythroid burst forming unit derived glycophorin+ cells. The activation of these signal transduction pathways was further correlated with various biological effects such as (i) cell proliferation, (ii) inhibition of apoptosis, (iii) activation of adhesion and (iv) secretion of the matrix metalloproteinases (MMPs) MMP‐9 and MMP‐2, and vascular endothelial growth factor (VEGF). We found that in human CD34+ cells and erythroblasts erythropoietic factors may activate similar but different signalling pathways, and that activation of each of the JAK‐STAT, MAPK p42/44 or PI‐3K‐AKT axes alone is not sufficient either to stimulate cell proliferation or inhibit apoptosis, suggesting that these processes are regulated by orchestrated activation of multiple signalling cascades. Accordingly, we found that although cell proliferation was more related to simultaneous activation of JAK‐STAT and MAPK p42/44, the effect on cell survival correlated with activation of PI‐3K‐AKT, MAPK p42/44 and JAK‐STAT proteins. We also demonstrated that differentiating normal human erythroid cells lose their adhesive properties and secrete angiopoietic factors such as MMP‐9, MMP‐2 and VEGF, and we postulate that this secretion by early erythroid cells may play a role in their maturation and egress from the haematopoietic niches of the bone marrow.

In vitro expansion of human megakaryocytes as a tool for studying megakaryocytic development and function

Research on normal human megakaryopoiesis has been limited by technical problems in obtaining megakaryocytic cells in sufficient quantities for experimental purposes. We describe here an ex vivo serum-free liquid culture system to expand normal human megakaryoblasts from purified bone marrow-, cord blood- or peripheral blood-derived CD34(+) cells. The early megakaryocytic cells are expanded in the presence of recombinant thrombopoietin (TpO) and interleukin-3 (IL-3), and if necessary further purified by employing anti-CD61 immunomagnetic beads. Our expansion system generates normal human megakaryoblasts in quantities sufficient to perform various functional studies on these cells as well as to isolate from them proteins and mRNA for molecular analysis. Megakaryocytic cells isolated from these cultures (i) express several markers characteristic of this lineage (CD41, CD61, CD62 P, CXCR-4, PAR-1, etc.), (ii) respond by calcium flux and phosphorylation of various intracellular proteins to stimulation by thrombin and (iii) adhere to fibrinogen and vitronectin. However, human megakaryoblasts derived from the cultures supplemented with TpO + IL-3, in contrast to murine megakaryocytic cells cultured under similar conditions, display poor polyploidization and do not release platelets. Since IL-3 has been reported to inhibit final maturation of megakaryocytic cells, we recently modified our expansion strategy. In this new approach CD34(+) cells are first expanded for 11 days in the presence of TpO + IL-3. Then megakaryoblasts derived are expanded for an additional 7 days supplemented with TpO only. We found that megakaryocytic cells expanded in this two step culture model are more differentiated, are polyploid and release platelets. The model described here provides normal human megakaryoblasts in adequate numbers, to study megakaryopoiesis and megakaryocyte function.Research on normal human megakaryopoiesis has been limited by technical problems in obtaining megakaryocytic cells in sufficient quantities for experimental purposes. We describe here an ex vivo serum-free liquid culture system to expand normal human megakaryoblasts from purified bone marrow-, cord blood- or peripheral blood-derived CD34 + cells. The early megakaryocytic cells are expanded in the presence of recombinant thrombopoietin (TpO) and interleukin-3 (IL-3), and if necessary further purified by employing anti-CD61 immunomagnetic beads. Our expansion system generates normal human megakaryoblasts in quantities sufficient to perform various functional studies on these cells as well as to isolate from them proteins and mRNA for molecular analysis. Megakaryocytic cells isolated from these cultures (i) express several markers characteristic of this lineage (CD41, CD61, CD62 P, CXCR-4, PAR-1, etc.), (ii) respond by calcium flux and phosphorylation of various intracellular proteins to stimulation by thrombin and (iii) adhere to fibrinogen and vitronectin. However, human megakaryoblasts derived from the cultures supplemented with TpO + IL-3, in contrast to murine megakaryocytic cells cultured under similar conditions, display poor polyploidization and do not release platelets. Since IL-3 has been reported to inhibit final maturation of megakaryocytic cells, we recently modified our expansion strategy. In this new approach CD34 + cells are first expanded for 11 days in the presence of TpO + IL-3. Then megakaryoblasts derived are expanded for an additional 7 days supplemented with TpO only. We found that megakaryocytic cells expanded in this two step culture model are more differentiated, are polyploid and release platelets. The model described here provides normal human megakaryoblasts in adequate numbers, to study megakaryopoiesis and megakaryocyte function.

HSP90 antagonist, geldanamycin, inhibits proliferation, induces apoptosis and blocks migration of rhabdomyosarcoma cells in vitro and seeding into bone marrow in vivo.

In this study, geldanamycin (GA) was found to have an antiproliferative effect on both embryonal and alveolar rhabdomyosarcoma (RMS) cell lines. The maximum level of inhibition reached 80% for both embryonal and alveolar RMS. After GA treatment, cells also became apoptotic as judged by Annexin V-positive staining, activation of caspase-3 pathway and poly(ADP ribose) polymerase cleavage. GA was responsible for the arrest of RMS cells in both G1 and G2/M phases of the cell cycle. G1 blockade, however, was transient and was seen only in the first 24u2009h of GA treatment. RMS often gives distant metastases to various organs including bone marrow. RMS cells express high levels of MET receptor and respond to hepatocyte growth factor with increased motility. In our study, we found that GA decreased the level of MET expression and inhibited the chemotaxis of RMS cells toward the hepatocyte growth factor gradient. GA also blocked the homing of RMS cells into bone marrow of severe combined immune deficient mice. In all our experiments embryonal RMS cell lines were significantly more sensitive, and lower concentrations of GA were sufficient to block embryonal RMS cell proliferation, induce apoptosis and inhibit motility. Our data show that the HSP90 inhibitor GA has the potential to become a new drug in RMS treatment. It blocks RMS proliferation, decreases cell survival and inhibits motility of RMS cells.

ORIGINAL ARTICLE: The Characterization of the Subpopulation of Suppressive B7H4+ Macrophages and the Subpopulation of CD25+ CD4+ and FOXP3+ Regulatory T-cells in Decidua during the Secretory Cycle Phase, Arias Stella Reaction, and Spontaneous Abortion – A Preliminary Report

Problemu2002 The presence of immunosuppressive cells within the endometrium and decidua is crucial for establishing maternal immune tolerance against fetal antigens. We decided to evaluate the subpopulations of Treg cells and B7H4 macrophages in eutopic endometrium typified by Arias Stella reaction during the development of Fallopian tube pregnancy as well as in decidua at the time of spontaneous abortion (SA), and to compare these findings to those observed in the endometrium during the secretory cycle phase of healthy women.

Inhibition of Rhabdomyosarcoma's metastatic behavior through downregulation of MET receptor signaling

Rhabdomyosarcoma (RMS) is a soft tissue sarcoma usually diagnosed in children. In advanced and metastatic stages the prognosis is often poor. RMS cell lines were used for evaluation of the role of MET receptor inhibition on chemotaxis and invasion. In vivo studies were performed using NOD-SCID xenograft model. This study shows that blocking of MET expression has strong influence on metastatic behavior of RMS. MET negative cells possess a reduced potential to migrate and to invade. Downregulation of MET suppressed the ability of RMS cells to populate bone marrow. Inhibition of MET negative tumor cells engraftment into bone marrow was observed. MET negative tumors were also two to four times smaller than their wild type counterparts. Since MET receptor plays a very important role in facilitating metastasis of RMS cells, blocking of HGF-MET axis might be considered as a therapeutic option for RMS patients, at more advanced and metastatic stages.

Heparinized cadaveric organ donors (HCOD)--a potential source of hematopoietic cells for transplantation and gene therapy.

Background. Hematopoietic stem cells (HSC) from unrelated HLA-matched heparinized cadaveric organ donors (HCOD) are a new potential source of cells for transplantation and gene therapy. In addition, these cells could also be used as adjuvant therapy to increase microchimerism and graft tolerance after transplantations of various solid organs. Our purpose was to develop an efficient method for harvesting hematopoietic cells from HCODs, Methods. Bone marrow cells were harvested from pelvic bones and/or vertebral bodies from 50 adult HCODs before or up to 3 hr after disconnecting the donor from the respirator. Subsequently, we evaluated the hematological and gasometric parameters of aspirated marrow samples as well as the proliferative potential, viability, and expression of CD34 and AC133 antigens on these cells. Results. We noticed that up to 2–3 hr after disconnecting the donor from the respirator bone marrow cavities do not clot and remain uninfected and that it is possible to aspirate bone marrow mononuclear cells in quantities sufficient to perform allotransplantation. Nevertheless, due to the developing hypoxia and acidosis of the hematopoietic microenvironment the number and proliferative potential of CD34+ and AC133+ cells gradually decreases. Hence, to obtain viable early hematopoietic cells, bone marrow should be aspirated without delay; optimally before HCOD is disconnected from the respirator or at the very latest 2 hr after organ harvest. Conclusions. Collectively, our results show that early hemopoietic cells may be efficiently harvested from HCOD in large quantities and used for research and/or transplantation purposes. We postulate to create an international network of banks in which hemopoietic stem cells from HCODs could be preserved for therapeutic purposes.

Biological significance of the different erythropoietic factors secreted by normal human early erythroid cells.

Evidence is accumulating that autocrine/paracrine regulatory mechanisms play an important role in regulating normal hematopoiesis. To support this, various growth factors, cytokines and chemokines are expressed and secreted by normal early and differentiated hematopoietic cells. In this review, we summarize recent advances in the identification and understanding of the role of autocrine/paracrine axes in normal human erythropoiesis. We will also address a biological significance of the secretion of (i) metalloproteinases which in addition to growth factors and cytokines are secreted by normal erythroid cells and (ii) membrane-derived microvesicles (MV), that are shed from the surface of maturating erythroblasts/reticulocytes, and as we postulate may also play a role in intercellular communication. We hypothesize that all these factors together play an important role in a crosstalk between erythroid cells and their environment. A better understanding of intercellular crosstalk operating in normal erythropoiesis and of the mechanisms regulating synthesis of these endogenously produced factors may allow us to develop more efficient therapeutic strategies to treat various erythropoietic disorders.