Adhesion Of CD34 Research Articles

ACE2/Ang-(1-7)/Mas axis stimulates vascular repair-relevant functions of CD34+ cells.

CD34(+) stem/progenitor cells have been identified as a promising cell population for the autologous cell-based therapies in patients with cardiovascular disease. The counter-regulatory axes of renin angiotensin system, angiotensin converting enzyme (ACE)/Ang II/angiotensin type 1 (AT1) receptor and ACE2/Ang-(1-7)/Mas receptor, play an important role in the cardiovascular repair. This study evaluated the expression and vascular repair-relevant functions of these two pathways in human CD34(+) cells. CD34(+) cells were isolated from peripheral blood mononuclear cells (MNCs), obtained from healthy volunteers. Expression of ACE, ACE2, AT1, and angiotensin type 2 and Mas receptors were determined. Effects of Ang II, Ang-(1-7), Norleu(3)-Ang-(1-7), and ACE2 activators, xanthenone (XNT) and diminazene aceturate (DIZE) on proliferation, migration, and adhesion of CD34(+) cells were evaluated. ACE2 and Mas were relatively highly expressed in CD34(+) cells compared with MNCs. Ang-(1-7) or its analog, Norleu(3)-Ang-(1-7), stimulated proliferation of CD34(+) cells that was associated with decrease in phosphatase and tensin homologue deleted on chromosome 10 levels and was inhibited by triciribin, an AKT inhibitor. Migration of CD34(+) cells was enhanced by Ang-(1-7) or Norleu(3)-Ang-(1-7) that was decreased by a Rho-kinase inhibitor, Y-27632. In the presence of Ang II, XNT or DIZE enhanced proliferation and migration that were blocked by DX-600, an ACE2 inhibitor. Treatment of MNCs with Ang II, before the isolation of CD34(+) cells, attenuated the proliferation and migration to stromal derived factor-1α. This attenuation was reversed by apocynin, an NADPH oxidase inhibitor. Adhesion of MNCs or CD34(+) cells to fibronectin was enhanced by Ang II and was unaffected by Ang-(1-7). This study suggests that ACE2/Ang-(1-7)/Mas pathway stimulates functions of CD34(+) cells that are cardiovascular protective, whereas Ang II attenuates these functions by acting on MNCs. These findings imply that activation of ACE2/Ang-(1-7)/Mas axis is a promising approach for enhancing reparative outcomes of cell-based therapies.

Abstract 557: ACE2/Angiotensin-(1-7) Stimulates Vasoprotection-Relevant Functions of Human CD34 + Cells

Angiotensin converting enzyme 2 (ACE2) and angiotensin (Ang)-(1-7), key members of the vasoprotective axis of the renin-angiotensin system (RAS), have been shown to modulate hematopoietic functions of bone marrow-derived cells. This coupled with recent studies implicating the involvement of bone marrow stem/progenitor cells in the cardiovascular functions, we evaluated the expression of RAS components and functions of ACE2 and Ang-(1-7) in human CD34 + cells, hematopoietic stem cells with vasoprotective functions. Peripheral blood was obtained from healthy individuals and CD34 + cells were isolated by immunomagnetic separation. Real-time PCR was carried out, and the effects of Ang II, Ang-(1-7), NorLeu 3 Ang-(1-7), and ACE2 activators, diminazene aceturate (DIZI) and a xanthenone derivative (XNT), were evaluated on migration, proliferation and adhesion of CD34 + cells. Paracrine factors produced by CD34 + cells were analyzed. CD34 + cells showed the presence of mRNA transcripts of ACE, ACE2 and Mas1 receptor. In contrast, renin, angiotensinogen, and AT1 and AT2 receptors were not detectable. Treatment with 100nM Ang-(1-7) or 10nM NorLeu 3 Ang-(1-7) stimulated proliferation and migration of CD34 + cells while adhesion of these cells to fibronectin was not affected. Ang-(1-7) treatment altered the paracrine profile of CD34 + cells: increased IL10, IL8, G-CSF, GM-CSF, IGFBP-3 and decorin (P<0.03), and decreased IGF1, thrombopoietin and TGFβ1 levels (P<0.05, n=5). In contrast, 100nM Ang II has no effects on proliferation and migration of CD34 + cells. However, in the presence of Ang II, 100nM DIZI or 100nM XNT increased proliferation and migration of CD34 + cells compared to the cells treated with Ang II alone (P<0.05, n=7). Treatment of total leucocytes with Ang II prior to the isolation of CD34 + cells attenuated their migration to stromal-derived factor-1α and proliferation compared to the untreated (P<0.05, n=5), and enhanced their adhesion to fibronectin. These studies suggest that activation of endogenous ACE2 or treatment with Ang-(1-7) stimulates the functions of CD34 + cells that are indicators of their cardiovascular protective functions while Ang II may attenuate these properties indirectly via acting on leucocytes.

Imatinib and Nilotinib Inhibit Hematopoietic Progenitor Cell Growth, but Do Not Prevent Adhesion, Migration and Engraftment of Human Cord Blood CD34+ Cells

BackgroundThe availability of tyrosine kinase inhibitors (TKIs) has considerably changed the management of Philadelphia chromosome positive leukemia. The BCR-ABL inhibitor imatinib is also known to inhibit the tyrosine kinase of the stem cell factor receptor, c-Kit. Nilotinib is 30 times more potent than imatinib towards BCR-ABL in vitro. Studies in healthy volunteers and patients with chronic myelogenous leukemia or gastrointestinal stromal tumors have shown that therapeutic doses of nilotinib deliver drug levels similar to those of imatinib. The aim of this study was to compare the inhibitory effects of imatinib and nilotinib on proliferation, differentiation, adhesion, migration and engraftment capacities of human cord blood CD34+ cells.Design and MethodsAfter a 48-hour cell culture with or without TKIs, CFC, LTC-IC, migration, adhesion and cell cycle analysis were performed. In a second time, the impact of these TKIs on engraftment was assessed in a xenotransplantation model using NOD/SCID/IL-2Rγ (null) mice.ResultsTKIs did not affect LTC-IC frequencies despite in vitro inhibition of CFC formation due to inhibition of CD34+ cell cycle entry. Adhesion of CD34+ cells to retronectin was reduced in the presence of either imatinib or nilotinib but only at high concentrations. Migration through a SDF-1α gradient was not changed by cell culture in the presence of TKIs. Finally, bone marrow cellularity and human chimerism were not affected by daily doses of imatinib and nilotinib in a xenogenic transplantation model. No significant difference was seen between TKIs given the equivalent affinity of imatinib and nilotinib for KIT.ConclusionsThese data suggest that combining non-myeloablative conditioning regimen with TKIs starting the day of the transplantation could be safe.

Maturing EPCs into endothelial cells: may the force be with the EPCs. Focus on “Fluid shear stress induces differentiation of circulating phenotype endothelial progenitor cells”

endothelial progenitor cells (EPCs) are a unique cell type found circulating in the peripheral blood with the capacity to become mature endothelial cells. EPCs can be released from many sources including the bone marrow, adipose tissue, the vessel wall, as well as potentially the spleen, liver, and

The Disease-Related Bone Marrow Microenvironment Alters Hematopoietic Stem and Progenitor Function in Multiple Myeloma Patients

Abstract 2898Multiple myeloma (MM) patients often suffer from hematopoietic impairment already at the time of diagnosis with anemia as the prevailing symptom. Given the overt affection of the bone marrow in MM patients by the invasion of malignant plasma cells, we hypothesized that hematopoietic insufficiency in these patients may originate from a functional impairment of hematopoietic stem and progenitor cells.Quantitative analysis of BM CD34+ HSPC cell subsets from MM patients and age-matched healthy donors showed a significant decline of all HSPC subsets including hematopoietic stem cells, common myeloid and lymphoid progenitors, granulocyte-macrophage progenitors and megakaryocyte-erythrocyte progenitors in MM patients. The greatest diminution was observed in megakaryocyte-erythrocyte progenitors (MEP) which were 4.9-fold reduced in comparison to healthy donors.Transcriptional analyses of CD34+ HSPC subsets revealed a significant deregulation of signaling pathways that was particularly striking for TGF beta signaling and suggested increased activation of this signaling pathway. Immunhistochemical staining of phosphorylated smad2, the downstream mediator of TGF receptor I kinase activation, in bone marrow sections and immunoblotting of purified CD34+ HSPC of MM patients confirmed the overactivation of TGF beta signaling.On a functional level, we observed significantly reduced long-term self-renewal and clonogenic growth, particularly of the erythroid precursors BFU-E and CFU-E, in CD34+ HSPC of MM patients which could be restored by inhibition of TGF beta signaling. Proliferation and cell cycle analyses revealed a significantly decreased proliferation activity in CD34+ HSPC and, particularly, MEP. Again, this was reversible after inhibition of TGF beta signaling.In addition, the transcriptional analyses showed disturbance of pathways involved in the adhesion and migration of HSPC and the gene encoding for the principal hyaluronan receptor CD44 throughout the HSPC subsets. This was corroborated by immunofluorescence imaging of CD44 on HSPC subsets showing a marked downregulation in the patients' cells. In line, the adhesion of CD34+ HSPC subsets to hyaluronan and their migration towards SDF-1 was significantly inhibited.Subsequent xenotransplantation of CD34+ HSPC from MM patients and healthy donors into myeloma-free recipients revealed even increased long-term engraftment of CD34+ HSPC obtained from MM patients and normal differentiation capacities suggesting that the observed functional alterations in fact depend on the MM-related bone marrow microenvironment.Our data show that hematopoietic impairment in patients with multiple myeloma originates, at least in part, from functional alterations of hematopoietic stem and progenitor cells. These alterations seem to depend on the disease-related changes of the bone marrow microenvironment. Currently, experiments are underway to elucidate in more detail the role of the microenvironment and the responsible structures for the impairment of HSPC in MM patients. These data will be presented. Disclosures:Kobbe:Celgene: Consultancy, Research Funding; Ortho Biotec: Consultancy.

Differential chemokine receptor expression regulates functional specialization of endothelial progenitor cell subpopulations

Postnatal vasculogenesis is mediated by endothelial progenitor cells (EPCs) which consist of subpopulations with different functional capacities. Our goal was to profile chemokine receptor expression on relevant subsets of EPCs and to characterize their role for effector functions. CD34(+)/CD133(+)/VEGFR2(+) EPCs were characterized by high expression of chemokine receptors CXCR4, CX3CR1, BLT1, and low level expression of CXCR2 and CCR2, while primordial CD34(-)/CD133(+)/VEGFR2(+) EPCs express these chemokine receptors at comparably low levels. Migration assays revealed that SDF-1, fractalkine, and LTB4 significantly increase migration of CD34(-)/CD133(+)/VEGFR2(+) EPCs, while SDF-1 was the only potent agonist of migration of CD34(+)/CD133(+)/VEGFR2(+) EPCs. SDF-1, fractalkine, and LTB4 trigger significant increase adhesion of CD34(+)/CD133(+)/VEGFR2(+) EPCs, while in CD34(-)/CD133(+)/VEGFR2(+) EPCs SDF-1 and fractalkine are equipotent agonists and LTB4 triggers a smaller though still significant increase in adhesion. Differential expression of specific chemokine receptors is an important regulator in terms of migration and adhesion of biologically relevant EPC-subpopulations, which may have implications for cell therapeutic strategies for treatment of ischemic vascular disease.

βig-h3, a Novel Regulator of Adhesion and Differentiation of Human Hematopoietic Stem/Progenitor Cells.

Abstract 3640Poster Board III-576Adult hematopoietic stem cells (HSC) reside in the bone marrow (BM) in so-called niches. Within this specialized microenvironment, the interactions of HSC with adhesion molecules on neighbouring cells and extracellular matrix (ECM) components are thought to be critical for the maintenance of the HSC population.Comparative gene-expression profiling of purified HSC in homeostatic and regenerative conditions allowed the identification of a set of differentially expressed ECM proteins. One of these proteins was the novel ECM protein βg-h3, which plays a role in cell-ECM interactions, by binding to type I, II and IV collagens and cellular integrins. We postulated that βig-h3 could have a role in HSC biology by being both a homeostatic and regenerative regulator of HSC self-renewal and differentiation.First we analyzed the mRNA expression in human CD34+ hematopoietic stem/progenitor cells (HSPC) isolated from BM, mobilized peripheral blood (MPB) and umbilical cord blood (UCB). The expression of βig-h3 was found to be significantly higher in BM-CD34+ cells as compared to MPB-CD34+ cells, suggesting a role for this ECM protein in retaining HSC in the BM. To determine expression of βig-h3 on the various subsets within the heterogeneous CD34+ population, the expression was compared between sorted sub-populations of BM-CD34+ cells: megakaryocyte-erythrocyte-progenitors (MEP: CD38+/CD110+/CD45RA−), common myeloid progenitors (CMP: CD38+/CD110−/CD45RA−), granulocyte-monocyte-progenitors (GMP: CD38+/CD110−/CD45RA+) and more immature CD34+/CD38− HSC. The purity of the sub-populations was analyzed by colony forming assays. These data indicate that at least the mRNA expression of βig-h3 was highest in GMPs. Analysis of different human cell types revealed that the highest βig-h3 mRNA expression is measured in monocytes, dendritic cells and mesenchymal stromal cells (MSC), while its expression in megakaryocytes and HUVEC is comparable to that in HSPC. In addition, cell surface expression of the βig-h3 protein was determined by flowcytometry. βig-h3 was found to be expressed on the cell surface of only a subpopulation of BM derived CD34+ cells (0.5%), monocytes (5%), MSCs (11%) and megakaryocytes (30%). Intracellular flowcytometry staining revealed that βig-h3 is expressed inside CD34+ cells derived from all sources.Since there is evidence in several other cell types that βig-h3 plays a role in enhancing cell adhesion and migration, adhesion experiments using CD34+ cells were performed. These experiments show a significant (p<0.01) two-fold increased adhesion of MPB-CD34+ cells to βig-h3 compared to a BSA coating (mean 40% (SEM ± 9.8%) and 23% (SEM ± 5.0%), respectively, (n=3)). Further experiments showed that adhesion of CD34+ cells to βig-h3 is mediated by both β1- and β2- integrins.The functional relevance of the target proteins in HSC differentiation and self-renewal was studied by lentiviral mediated overexpression. We used a βig-h3-SIN-GFP vector or a control SIN-GFP vector to transduce CD34+ cells isolated from MPB or UCB and cultured them towards a megakaryocytic lineage using TPO, SCF, Flt3 and IL6. Overexpression of βig-h3 in MPB and UCB-CD34+ cells resulted in an acceleration of the megakaryopoiesis and in an increased percentage of mature megakaryocytic cells (i.e. CD41+) two weeks after transduction.In conclusion, βig-h3 is an adhesive protein for HSPCs and GMP's express significantly more βig-h3 as compared to other CD34+ subsets. Moreover, ectopic expression of βig-h3 in CD34+ cells accelerates differentiation towards megakaryocytes. These data suggest that upregulation of βig-h3 in HSCs may be a vital element driving lineage commitment of HSCs in homeostatic or regenerative conditions. Disclosures:No relevant conflicts of interest to declare.

The G Protein-Coupled Receptor cysLT1 Mediates Gi Protein-Dependent Chemotaxis and Adhesion of CD34+ Cells in Vitro Similar to CXCR4, but Not Progenitor Mobilization in Vivo.

CysLT1, which is expressed in several cell types during inflammation and allergy, and the homing related chemokine receptor CXCR4 both belong to the family of G protein-coupled receptors (GPCR). Similar to CXCR4, the cysteinyl-leukotriene receptor cysLT1 is strongly expressed in CD34+ cell lines and CD34+ hematopoietic progenitor cells (HPC). We therefore compared the effects mediated by cysLT1 on HPC to those observed after activation of CXCR4. The most potent cysLT1 ligand LTD4 induced chemotaxis and adhesion of CD34+ HPC to endothelial cells, immobilized VCAM-1 and fibronectin, which was comparable to effects induced by the CXCR4 ligand SDF-1. CysLT1- and CXCR4-mediated effects were inhibited by pertussis toxin (PTX), suggesting that both GPCRs employ the same Gi-protein-dependent signaling pathways in CD34+ HPC. This is supported by identical time courses of intracellular calcium fluxes and actin polymerization induced by LTD4 and SDF-1, as measured by time-dependent flow cytometry. Given the striking similarities of cysLT1- and CXCR4-mediated effects in vitro, one might expect also overlapping functions in vivo. We therefore investigated whether blocking of cysLT1 is associated with HPC mobilization. As cysLT1 antagonists are established for therapy and prophylaxis in patients with allergic and exercise-induced asthma, circulating CD34+ progenitors were enumerated after initiation of a treatment with the cysLT1 antagonist montelukast, used as asthma prophylaxis in otherwise healthy subjects. The number of CD34+ cells or white blood counts did not differ significantly from the baseline value 2, 4, 8, 12, and 24 hours after administration of 10 mg montelukast, in contrast to previous studies analyzing stem cell mobilization induced by CXCR4 antagonists. This corresponds with our in vitro findings that LTD4 is produced by bone marrow endothelium and stromal cells only when deprived of hematopoietic cells, in contrast to the constitutive production of SDF-1. We conclude that cysLT1 is not involved in bone marrow retention of HPC during steady-state hematopoiesis, but may modulate HPC homing when its ligands are produced either locally (i.e. bone marrow aplasia) or systemically (i.e. inflammation).

Abstract 5901: Cardiac Expression of CCN1, Capable of Promoting Adhesion of CD34+ Progenitor Cells, Is Strongly Induced in Parvovirus B19-Associated Cardiomyopathy

The clinical phenotype of human dilated cardiomyopathy (DCM) encompasses a broad spectrum of etiologically distinct disorders. We obtained the cardiac expression profile of a DCM subtype (DCMi-B19) characterized by cardiac inflammation and parvovirus B19 (B19) persistence, to identify possible new therapeutic targets in humans. By microarray analysis and TaqMan PCR endomyocardial biopsies from DCMi-P19 patients (n=8) were compared to controls w/o cardiac disease. In DCMi-B19 an expression profile with dysbalance between several gene regulatory networks was observed. Tab. 1/2 classify deregulated genes in a group closely correlated (p&lt;0.01) with left ventricular ejection fraction (EF) and another one w/o correlation to EF. CCN1 was the most strongly upregulated of the EF-independent genes which encompass further members of the CCN1 regulatory network including CCN2=CTGF. In contrast the genes in tab. 2 are closely EF-correlated. We have previously shown that CCN1 is induced in endothelial cells by pro-inflammatory cytokines in vitro, which may also be the mechanism of its induction in human DCMi-B19 hearts. Two recent studies described the potential of CCN1 to promote migration/adhesion of CD34+ stem cells, and of activated monocytes via alphaMbeta2 integrin. This suggests that the CCN1 induction in DCMi-B19 may influence the influx of CD34+ stem cells and activated monocytes to the inflamed heart, and obviously this may occur independent of hemodynamic impairment. B19 viruses infect cardiac microvascular endothelium, and endothelial CCN1 induction may therefore be involved in disease pathogenesis by altering circulating cell interactions with the inflamed heart. Table 1: EF-Independent Gene Deregulation Table 2: EF-Correlated Gene Deregulation

Protein Phosphatase 2A Plays an Important Role in Stromal Cell-Derived Factor-1/CXC Chemokine Ligand 12-Mediated Migration and Adhesion of CD34+ Cells

Migration of hemopoietic stem and progenitor cells (HSPC) is required for homing to bone marrow following transplantation. Therefore, it is critical to understand signals underlying directional movement of HSPC. Stromal cell-derived factor-1 (SDF-1)/CXCL12 is a potent chemoattractant for HSPC. In this study, we demonstrate that the serine-threonine protein phosphatase (PP)2A plays an important role in regulation of optimal level and duration of Akt/protein kinase B activation (a molecule important for efficient chemotaxis), in response to SDF-1. Inhibition of PP2A, using various pharmacological inhibitors of PP2A including okadaic acid (OA) as well as using genetic approaches including dominant-negative PP2A-catalytic subunit (PP2A-C) or PP2A-C small interfering RNA, in primary CD34(+) cord blood (CB) cells led to reduced chemotaxis. This was associated with impairment in polarization and slower speed of movement in response to SDF-1. Concomitantly, SDF-1-induced Akt phosphorylation was robust and prolonged. Following SDF-1 stimulation, Akt and PP2A-C translocate to plasma membrane with enhanced association of PP2A-C with Akt observed at the plasma membrane. Inhibition of PI3K by low-dose LY294002 partially recovered chemotactic activity of cells pretreated with OA. In addition to chemotaxis, adhesion of CD34(+) cells to fibronectin was impaired by OA pretreatment. Our study demonstrates PP2A plays an important role in chemotaxis and adhesion of CD34(+) CB cells in response to SDF-1. CD34(+) CB cells pretreated with OA showed impaired ability to repopulate NOD-SCID mice in vivo, suggesting physiological relevance of these observations.

Role of glycosylphosphatidylinositol-specific phospholipase D in the homing of umbilical cord blood, mobilized peripheral blood and bone marrow-derived hematopoietic stem/progenitor cells

Recent studies suggested that glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) correlated with tumor malignancy and prognosis of certain tumors. As hematopoietic stem/progenitor cells (HS/PC) homing was similar to tumor invasion and metastasis in some mechanisms, which arose our intrests in whether GPI-PLD contribution to the homing of HS/PC. In this study, CD34 + cells from umbilical cord blood (UCB), mobilized peripheral blood (MPB), and bone marrow (BM) were assayed for their differences in adhesion, migration, respectively. The expression of GPI-anchored proteins (CD48, CD90) on the cells were analyzed by flow cytometry. Semi-quantitive RT-PCR was used to detect GPI-PLD expression in the three different CD34 + cells. The results showed that GPI-PLD had no effect on the adhesion of CD34 + cells. While, spontaneous and SDF-1 induced migration of UCB and MPB, but not BM CD34 + cells were decreased after 1,10-phenanthroline (an inhibitor of GPI-PLD) pretreatment. Furthermore, we found little difference in GPI-anchored adhesion molecules (CD48, CD90) expression between untreated and pretreated CD34 + cells. GPI-PLD mRNA was low expressed in MPB and undetected in UCB and BM CD34 + cells. Our results suggested that GPI-PLD probably had no contribution to HS/PC homing, which may due to its low or no expression in UCB, BM and MPB CD34 + cells.

Protein Phosphatase2A Regulates Stromal Cell- Derived Factor-1 Mediated Responses of CD34+ Cord Blood Cells.

The chemokine stromal cell- derived factor-1(SDF-1) and its receptor, CXCR4, play a major role in migration, retention, and development of hematopoietic progenitors in the bone marrow. During steady-state hematopoiesis, CXCR4/SDF-1 interaction restricts hematopoietic stem progenitor cells in marrow and disruption of the SDF-1/CXCR4 axis leads to their egress into circulation. However, biological responses to SDF-1 are rapidly attenuated by overlapping mechanisms that include peptide degradation by cell surface proteases, receptor uncoupling from heterotrimeric G-proteins and receptor endocytosis. It is known that phosphorylation of G-protein coupled receptors (GPCRs) by several protein kinases including serine-threonine kinase, protein kinase C (PKC), can result in receptor desensitization. This prompted us to investigate whether the role of serine- threonine phosphatase in the regulation of SDF-1 induced responses of CD34+ cord blood (CB) cells. To investigate the role of serine- threonine phosphatase we evaluated the effect of okadaic acid, a specific serine-threonine phosphatase inhibitor, on SDF-1 induced chemotaxis and adhesion of CD34+ cells from cord blood. Pre-incubation of CD34+ CB cells with okadaic acid (100–1000nM) significantly reduced SDF-1 directed chemotaxis and adhesion of CD34+ CB cells. This correlated with an increase in PKC phosphorylation. Although primary SDF-1 induced calcium flux in CD34+CB cells was unaffected by okadaic acid, if the cells were pretreated with SDF-1 and then stimulated with SDF-1, calcium flux was significantly less in CD34+CB cells pretreated with okadaic acid compared to untreated cells. Further, confocal microscopy was used to demonstrate the co-localization of serine threonine phosphatase and PKC. Using antibodies specific to PP2A catalytic subunit (PP2Ac) and various classes of PKC super family, it was observed in unstimulated CD34+ CB cells PP2Ac was mainly localized in nucleus and some phosphor-PKC-delta was detected in the cytoplasm. However, upon SDF-1 stimulation, co-localization of PP2Ac and phospho-PKC-delta was observed on the membrane of CD34+CB cells. These results indicate that serine-threonine phosphatase regulate SDF-1 mediated responses in CD34+CB cells and may play an important role in the ability of hematopoietic stem progenitor cells to continually respond to SDF-1.

Atypical PKC-ζ regulates SDF-1–mediated migration and development of human CD34+ progenitor cells

The chemokine stromal cell–derived factor–1 (SDF-1) and its receptor, CXCR4, play a major role in migration, retention, and development of hematopoietic progenitors in the bone marrow. We report the direct involvement of atypical PKC-ζ in SDF-1 signaling in immature human CD34+-enriched cells and in leukemic pre-B acute lymphocytic leukemia (ALL) G2 cells. Chemotaxis, cell polarization, and adhesion of CD34+ cells to bone marrow stromal cells were found to be PKC-ζ dependent. Overexpression of PKC-ζ in G2 and U937 cells led to increased directional motility to SDF-1. Interestingly, impaired SDF-1–induced migration of the pre-B ALL cell line B1 correlated with reduced PKC-ζ expression. SDF-1 triggered PKC-ζ phosphorylation, translocation to the plasma membrane, and kinase activity. Furthermore we identified PI3K as an activator of PKC-ζ, and Pyk-2 and ERK1/2 as downstream targets of PKC-ζ. SDF-1–induced proliferation and MMP-9 secretion also required PKC-ζ activation. Finally, we showed that in vivo engraftment, but not homing, of human CD34+-enriched cells to the bone marrow of NOD/SCID mice was PKC-ζ dependent and that injection of mice with inhibitory PKC-ζ pseudosubstrate peptides resulted in mobilization of murine progenitors. Our results demonstrate a central role for PKC-ζ in SDF-1–dependent regulation of hematopoietic stem and progenitor cell motility and development.

Atypical PKC-ζ regulates SDF-1–mediated migration and development of human CD34+ progenitor cells

The chemokine stromal cell-derived factor-1 (SDF-1) and its receptor, CXCR4, play a major role in migration, retention, and development of hematopoietic progenitors in the bone marrow. We report the direct involvement of atypical PKC-zeta in SDF-1 signaling in immature human CD34(+)-enriched cells and in leukemic pre-B acute lymphocytic leukemia (ALL) G2 cells. Chemotaxis, cell polarization, and adhesion of CD34(+) cells to bone marrow stromal cells were found to be PKC-zeta dependent. Overexpression of PKC-zeta in G2 and U937 cells led to increased directional motility to SDF-1. Interestingly, impaired SDF-1-induced migration of the pre-B ALL cell line B1 correlated with reduced PKC-zeta expression. SDF-1 triggered PKC-zeta phosphorylation, translocation to the plasma membrane, and kinase activity. Furthermore we identified PI3K as an activator of PKC-zeta, and Pyk-2 and ERK1/2 as downstream targets of PKC-zeta. SDF-1-induced proliferation and MMP-9 secretion also required PKC-zeta activation. Finally, we showed that in vivo engraftment, but not homing, of human CD34(+)-enriched cells to the bone marrow of NOD/SCID mice was PKC-zeta dependent and that injection of mice with inhibitory PKC-zeta pseudosubstrate peptides resulted in mobilization of murine progenitors. Our results demonstrate a central role for PKC-zeta in SDF-1-dependent regulation of hematopoietic stem and progenitor cell motility and development.

Human dendritic cells express neuronal Eph receptor tyrosine kinases: role of EphA2 in regulating adhesion to fibronectin

AbstractEph receptor tyrosine kinases and their ligands, the ephrins, have been primarily described in the nervous system for their roles in axon guidance, development, and cell intermingling. Here we address whether Eph receptors may also regulate dendritic cell (DC) trafficking. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that DCs derived from CD34+progenitors, but not from monocytes, expressed several receptors, in particular EphA2, EphA4, EphA7, EphB1, and EphB3 mRNA. EphB3 was specifically expressed by Langerhans cells, and EphA2 and EphA7 were expressed by both Langerhans- and interstitial-type DCs. EphA and EphB protein expression on DCs generated in vitro was confirmed by staining with ephrin-A3-Fc and ephrin-B3-Fc fusion proteins that bind to different Eph members, in particular EphA2 and EphB3. Immunostaining with anti-EphA2 antibodies demonstrated the expression of EphA2 by immature DCs and by skin Langerhans cells isolated ex vivo. Interestingly, ephrin expression was detected in epidermal keratinocytes and also in DCs. Adhesion of CD34+-derived DCs to fibronectin, but not to poly-l-lysine, was increased in the presence of ephrin-A3-Fc, a ligand of EphA2, through a β1integrin activation pathway. As such, EphA2/ephrin-A3 interactions may play a role in the localization and network of Langerhans cells in the epithelium and in the regulation of their trafficking. (Blood. 2003;102:4431-4440)

Laminin isoform–specific promotion of adhesion and migration of human bone marrow progenitor cells

AbstractLaminins are αβγ heterotrimeric extracellular proteins that regulate cellular functions by adhesion to integrin and nonintegrin receptors. Laminins containing α4 and α5 chains are expressed in bone marrow, but their interactions with hematopoietic progenitors are unknown. We studied human bone marrow cell adhesion to laminin-10/11 (α5β1γ1/α5β2γ1), laminin-8 (α4β1γ1), laminin-1 (α1β1γ1), and fibronectin. About 35% to 40% of CD34+ and CD34+CD38− stem and progenitor cells adhered to laminin-10/11, and 45% to 50% adhered to fibronectin, whereas they adhered less to laminin-8 and laminin-1. Adhesion of CD34+CD38− cells to laminin-10/11 was maximal without integrin activation, whereas adhesion to other proteins was dependent on protein kinase C activation by 12-tetradecanoyl phorbol-13-acetate (TPA). Fluorescence-activated cell-sorting (FACS) analysis showed expression of integrin α6 chain on most CD34+ and CD34+CD38−cells. Integrin α6 and β1 chains were involved in binding of both cell fractions to laminin-10/11 and laminin-8. Laminin-10/11 was highly adhesive to lineage-committed myelomonocytic and erythroid progenitor cells and most lymphoid and myeloid cell lines studied, whereas laminin-8 was less adhesive. In functional assays, both laminin-8 and laminin-10/11 facilitated stromal-derived factor-1α (SDF-1α)–stimulated transmigration of CD34+ cells, by an integrin α6 receptor–mediated mechanism. In conclusion, we demonstrate laminin isoform–specific adhesive interactions with human bone marrow stem, progenitor, and more differentiated cells. The cell-adhesive laminins affected migration of hematopoietic progenitors, suggesting a physiologic role for laminins during hematopoiesis.

Adhesion of CD34+ marrow precursors to human stroma is related to alphaSM actin expression by human marrow myofibroblasts.

We have studied the adhesion of human marrow CD34(+) precursors to stromal layer of nontransformed human marrow myofibroblasts (normal stroma) and to different stromal cell lines immortalized by T (PU-34) or t and T (L88/5, L87/4, L2Ori-, KM-102) oncogenes from simian virus 40 and E6, E7 oncogenes from human papilloma virus 16 (HS-27A, HS-23). Flow cytometry and Western blotting studies showed that cells from all lines were stromal myofibroblasts similar to normal stroma. Using an original method of adhesion measurement, we found that adhesion of CD34(+) cells was significantly increased on PU-34 cell layer as compared to normal stroma (43% vs. 27%) whereas adhesion on HS-27A and HS-23 was significantly decreased (11% and 8.5%, respectively), and adhesion on L88/5, L87/4, KM-102 and L2Ori- was negligible to nil (<6%). Adhesion of CD34(+) cells to stromal layers paralleled the expression of alpha smooth muscle (alphaSM) actin within the microfilaments of the cells from the different lines and was inversely correlated to their anchorage-independent growth in semisolid agar. These data show that adhesion to the stromal layer of CD34(+) cells is related to the alphaSM actin microfilamentous network in marrow myofibroblasts and that transformation can negatively affect this microfilamentous network and therefore adhesion of hematopoietic precursors.

Opposing effects of engagement of integrins and stimulation of cytokine receptors on cell cycle progression of normal human hematopoietic progenitors

We evaluated the effect of beta1-integrin receptor engagement on the expression and activity of cell cycle regulatory proteins in CD34(+) cells under conditions that mimic the steady-state marrow microenvironment and in the presence of supraphysiological concentrations of interleukin-3 (IL3) and stem cell factor (SCF). Adhesion of CD34(+) progenitors to fibronectin (FN) was similar whether IL3 or SCF was present or absent. Engagement of beta1-integrins blocked S-phase entry of CD34(+) cells in the absence of IL3 or SCF, whereas addition of 10 ng/mL IL3 or SCF prevented such a block in S-phase entry. In the absence of IL3 or SCF, cyclin-E levels were significantly lower and p27(KIP1) levels significantly higher in FN-adherent than in FN-nonadherent cells, or than in poly-L-lysine (PLL)-adherent or (PLL)-nonadherent cells. Cyclin-dependent-kinase (cdk)-2 activity was decreased and levels of cyclin-E-cdk2 complexes were lower in FN-adherent than in PLL-adherent cells. In contrast, cyclin-E and p27(KIP1) protein levels and cdk2 activity in cells adherent to FN in the presence of IL3 or SCF were similar to those in PLL-adherent and FN-nonadherent or PLL-nonadherent cells. In conclusion, under physiological cytokine conditions, integrin engagement prevents S-phase entrance of CD34(+) cells, which is associated with elevated levels of the contact-dependent cyclin kinase inhibitor p27(KIP1). Supraphysiological concentrations of IL3 or SCF prevent p27(KIP1) elevation and override the integrin-mediated inhibition of entry into S phase.

Alpha4beta1 integrin-mediated adhesion of CD34+ cells from patients with chronic myeloid leukaemia: influence of IL-3.

Interactions between integrins on haemopoietic progenitor cells and their stromal ligands have an important role in the control of haemopoiesis. Growth factors can modulate these interactions (so-called 'inside-out' signalling) resulting in changes in ligand binding activity. We have studied alpha4beta1 integrin-mediated adhesion to the H120 fragment of fibronectin (which contains the strongest alpha4beta1 binding site) in CD34+ cells from patients with chronic myeloid leukaemia (CML) and have determined the effect of IL-3 on the level of adhesion. Compared to normal CD34+ cells isolated from cord blood and peripheral blood progenitor harvests (mean of 61.4 +/- 14.9% of cells attached) the CML CD34+ cells showed reduced levels of adhesion (mean of 41.9 +/- 14.7%, P < 0.05). The effect of 10 ng/ml of IL-3 resulting in reduced adhesion of normal CD34+ cells at 30 min was absent in 6/7 patients with CML. Abnormalities of adhesion to fibronectin may thus be related to IL-3 pathways affected by BCR-ABL. These findings will have implications for understanding the dysregulation of growth and adhesion in CML.

Different Adhesive Characteristics and VLA-4 Expression of CD34+ Progenitors in G0/G1 Versus S+G2/M Phases of the Cell Cycle

We identified the cell cycle status of CD34+ cells of steady-state bone marrow (BM) and peripheral blood (PB) obtained from healthy volunteers, and those of apherasis PB samples collected from healthy donors who had been administered granulocyte colony-stimulating factor (G-CSF). More than 10% of CD34+ cells in BM were in S+G2/M phase. In contrast, regardless of whether G-CSF treatment was performed, less than 2% of CD34+ cells in PB were cycling. BM CD34+ cells showed greater VLA-4 expression and adherence to stromal cells than PB CD34+cells. In addition, when cycling and dormant BM CD34+cells were analyzed separately, the cells in S+G2/M phase expressed more VLA-4 and adhered to the stromal cell monolayer more efficiently than the cells in G0/G1 phase. Furthermore, this adhesion of CD34+ cells to the stromal cell layer was almost completely inhibited by anti-VLA-4 antibody. Taken together, these results suggest that CD34+ progenitors in G0/G1 phase of the cell cycle differ from those in S+G2/M phase in adhesiveness mediated by VLA-4 in the hematopoietic microenvironment.© 1998 by The American Society of Hematology.