Biomedical subjects

Thalia Papayannopoulou

Publications and source records attributed to Thalia Papayannopoulou.

At least 19 recordsLinked to original sources

PubMed2006

Sustained alterations in biodistribution of stem/progenitor cells in Tie2Cre+ alpha4(f/f) mice are hematopoietic cell autonomous.

We have generated Tie2Cre+alpha4(f/f) mice with documented alpha4-integrin ablation in hematopoietic and endothelial cells. A prominent feature in this model is a sustained, significant increase in circulating progenitors at levels higher than the levels seen with Tie2Cre+VCAM-1(f/f) mice. To test whether phenotypic differences are due to contributions by ligands other than VCAM-1 in bone marrow, or to alpha4-deficient endothelial cells or pericytes, we carried out transplantation experiments using these mice as donors or as recipients. Changes in progenitor biodistribution after transplantation were seen only with alpha4-deficient donor cells, suggesting that these cells were necessary and sufficient to reproduce the phenotype with no discernible contribution by alpha4-deficient nonhematopoietic cells. Because several similarities are seen after transplantation between our results and those with CXCR4-/- donor cells, the data suggest that VLA4/VCAM-1 and CXCR4/CXCL12 pathways contribute to a nonredundant, ongoing signaling required for bone marrow retention of progenitor cells during homeostasis.

Animals↗

PubMed2006

Definitive-like erythroid cells derived from human embryonic stem cells coexpress high levels of embryonic and fetal globins with little or no adult globin.

Human embryonic stem cells are a promising tool to study events associated with the earliest ontogenetic stages of hematopoiesis. We describe the generation of erythroid cells from hES (H1) by subsequent processing of cells present at early and late stages of embryoid body (EB) differentiation. Kinetics of hematopoietic marker emergence suggest that CD45+ hematopoiesis peaks at late D14EB differentiation stages, although low-level CD45- erythroid differentiation can be seen before that stage. By morphologic criteria, hES-derived erythroid cells were of definitive type, but these cells both at mRNA and protein levels coexpressed high levels of embryonic (epsilon) and fetal (gamma) globins, with little or no adult globin (beta). This globin expression pattern was not altered by the presence or absence of fetal bovine serum, vascular endothelial growth factor, Flt3-L, or coculture with OP-9 during erythroid differentiation and was not culture time dependent. The coexpression of both embryonic and fetal globins by definitive-type erythroid cells does not faithfully mimic either yolk sac embryonic or their fetal liver counterparts. Nevertheless, the high frequency of erythroid cells coexpressing embryonic and fetal globin generated from embryonic stem cells can serve as an invaluable tool to further explore molecular mechanisms.

Cell Differentiation↗

PubMed2006

The p67 laminin receptor identifies human erythroid progenitor and precursor cells and is functionally important for their bone marrow lodgment.

The laminins are a group of extracellular matrix proteins with constitutive expression in all tissues, including bone marrow stroma. A functional role for the nonintegrin laminin receptor p67 has been described for cancer metastasis and lymphocyte trafficking. Expression of p67 was also reported for other subsets of mature leukocytes and for malignant hematopoietic or nonhematopoietic cells. We explored p67 expression on normal hematopoietic progenitor cells (HPCs) and its putative role in bone marrow retention of transplanted HPCs. We found p67 expression on a subset of primary human CD34(+) cells coexpressing erythroid markers. Of importance, p67 recognizes early erythroid progenitors, since sorted p67(+) cells were significantly enriched for burst-forming units-erythroid (BFU-Es) and depleted of colony-forming units--granulocyte/macrophage (CFU-GMs). Blockade of p67 binding of donor cells, using antifunctional antibody, reduced bone marrow homing of BFU-Es. These studies identify p67 as a novel phenotypic marker for erythroid HPCs of functional importance for lineage-specific homing/retention among adult transplanted HPCs.

Adult↗

PubMed2006

Functional disruption of alpha4 integrin mobilizes bone marrow-derived endothelial progenitors and augments ischemic neovascularization.

The cell surface receptor alpha4 integrin plays a critical role in the homing, engraftment, and maintenance of hematopoietic progenitor cells (HPCs) in the bone marrow (BM). Down-regulation or functional blockade of alpha4 integrin or its ligand vascular cell adhesion molecule-1 mobilizes long-term HPCs. We investigated the role of alpha4 integrin in the mobilization and homing of BM endothelial progenitor cells (EPCs). EPCs with endothelial colony-forming activity in the BM are exclusively alpha4 integrin-expressing cells. In vivo, a single dose of anti-alpha4 integrin antibody resulted in increased circulating EPC counts for 3 d. In hindlimb ischemia and myocardial infarction, systemically administered anti-alpha4 integrin antibody increased recruitment and incorporation of BM EPCs in newly formed vasculature and improved functional blood flow recovery and tissue preservation. Interestingly, BM EPCs that had been preblocked with anti-alpha4 integrin ex vivo or collected from alpha4 integrin-deficient mice incorporated as well as control cells into the neovasculature in ischemic sites, suggesting that alpha4 integrin may be dispensable or play a redundant role in EPC homing to ischemic tissue. These data indicate that functional disruption of alpha4 integrin may represent a potential angiogenic therapy for ischemic disease by increasing the available circulating supply of EPCs.

Animals↗

PubMed2005

Anatomical compartments modify the response of human hematopoietic cells to a mitogenic signal.

Methods for specifically regulating transplanted cells have many applications in gene and cell therapy. We examined the response of human cord blood CD34+ cells to a specific mitotic signal in vivo. Using a conditional signaling molecule (F36VMpl) that is specifically activated by an artificial ligand called a chemical inducer of dimerization (CID), human hematopoietic cells transplanted into immune deficient mice were induced to proliferate. Only differentiating erythroid precursors and multipotential and erythroid progenitors (colony-forming unit [CFU]-mix and burst forming unitserythroid [BFUe]) responded; however, the nature of the response differed markedly between bone marrow and spleen. In the marrow, F36VMpl induced a 12- to 17-fold expansion of differentiated erythroid precursors and a loss of CFU-mix and BFUe. In the spleen, F36VMpl induced a marked rise in BFUe and CFU-mix and, relative to marrow, a much less prominent rise in more mature red cells. Clonal analysis was most consistent with the interpretation that the spleen and bone marrow differentially regulate the response of human progenitors to a mitotic signal, possibly influencing progenitor expansion versus differentiation. These findings establish CIDs as in vivo growth factors for human hematopoietic cells.

Animals↗

PubMed2005

Lack of alpha4 integrin expression in stem cells restricts competitive function and self-renewal activity.

Alpha4 integrin or VLA4 (CD49d/CD29) is a multitask molecule with wide expression within and outside the hematopoietic system. Because targeted ablation of alpha4 integrin leads to embryonic lethality, to study its effects on adult hematopoiesis, we used animals with conditional excision of alpha4 integrin (alpha4Delta/Delta) in hematopoietic cells. In such animals, we previously documented weakened bone marrow retention of progenitor cells during homeostasis and impaired homing and short-term engraftment after transplantation. In the present study we show that long-term repopulating cells lacking alpha4 integrins display a competitive disadvantage in hematopoietic reconstitution compared to normal competitors. Although initial dominance of alpha4+ competitors is due to their better homing and proliferative expansion early after transplantation, a progressive decline in contribution of alpha4Delta/Delta hematopoiesis is compatible with neither normal homing nor normal function of alpha4Delta/Delta hematopoietic stem cells (HSCs) in post-homing hematopoiesis. In the absence of alpha4+ competitor cells, alpha4Delta/Delta HSCs can establish long-term hematopoiesis in primary recipients, however, some resurgence of host hematopoiesis is evident, and it becomes dominant in secondary transplants, so that no survivors with exclusively alpha4Delta/Delta cells are seen in tertiary transplants. Collectively, our data provide compelling evidence that under regenerative stress alpha4 integrin assumes a greater importance than for maintenance of steady-state hematopoiesis.

Animals↗

PubMed2005

Hierarchy of molecular-pathway usage in bone marrow homing and its shift by cytokines.

Efficient bone marrow (BM) homing is a prerequisite for successful engraftment of transplanted hematopoietic cells (HPCs). Contradictory conclusions about the contribution of SDF-1/CXCR4 have clouded our understanding of its role within the molecular pathway cooperation needed for BM homing, particularly with the well-defined hierarchic network of adhesion molecules. In the present study we sought to unravel cooperative and compensatory molecular pathways guiding BM homing. Fresh BM-HPCs, rendered either SDF-1 unresponsive or Gi-signaling refractory, homed quite efficiently, because of compensation by alpha4-integrin interacting with VCAM-1. The contribution of SDF-1/CXCR4- or Gi-protein-mediated signals to BM homing became apparent after their blockade was combined with deletion of alpha4-integrin, leading to dramatic reduction in BM homing. Similar conclusions were revealed when VCAM-1-deficient hosts were used. Cytokine incubation changed the functional properties of BM-HPCs and hierarchy of molecular pathway usage in homing, by shifting the dominance among the homing mediators: loss of CXCR4 or Gi-signaling now significantly reduced BM homing, with only partial compensation through alpha4/VCAM-1 and endothelial selectins. These studies depict a flexible hierarchy of cooperating homing pathways, in which dominant players are repositioned with changing cytokine milieu, and possibly source of HPCs.

Animals↗

PubMed2005

VCAM-1 expression in adult hematopoietic and nonhematopoietic cells is controlled by tissue-inductive signals and reflects their developmental origin.

Although expression of vascular cell adhesion molecule 1 (VCAM-1) in endothelial cells and its functional implications have been previously appreciated, VCAM-1 expression in other than endothelial cells, especially hematopoietic cells, has been recently recognized and has not been explored in detail. Using normal mice and mice with a conditional ablation of VCAM-1 through a Tie2-driven cre transgene, we have studied the biodistribution and the pattern of VCAM-1 expression in circulating versus tissue-residing cells before and after their enforced mobilization. In the normal mouse, both at basal hematopoiesis or following mobilization, VCAM-1 expression is confined to myeloid cells residing in hematopoietic tissues, whereas free cells in circulation or in body cavities are devoid of VCAM-1 messenger RNA (mRNA) and protein. However, following culture, proliferating myeloid cells, but not lymphoid cells, express VCAM-1. In the VCAM-1-ablated mouse, there is an increase in circulating progenitors as a consequence of their ongoing release from bone marrow, a process enhanced by splenectomy. We postulate that the main mechanism leading to their release is the ablation of VCAM-1 by fibroblastic and by endothelial cells. Ablation of VCAM-1 in fibroblasts by Tie2-driven cre is a novel finding and likely denotes their developmental ancestry by Tie2-expressing (mesenchymal?) progenitor cells during development.

Age Factors↗

PubMed2005

Long-term functional impairment of hemopoietic progenitor cells engineered to express the S1 catalytic subunit of pertussis toxin.

OBJECTIVE: A large body of data suggests that pertussis toxin (PTX)-sensitive G protein signals in mature and immature hemopoietic cells control their migration patterns in vitro and in vivo. These effects were derived after treatment of cells or animals with PTX. To circumvent several inherent problems of PTX holotoxin treatment, we expressed the S1 catalytic activity of PTX, thus blocking Gi protein signaling, in 32D murine myeloid progenitor cells and in primary human CD34+ cells, and studied its functional consequences. METHODS: S1 was expressed using viral vectors. Effects of Gi protein blockade on proliferation, migration, adhesion, and gene expression were tested in vitro. RESULTS: S1 expression was nontoxic for the cells; expression and function were stable long-term and not overridden by compensatory mechanisms. S1-transduced 32D cells and primary CD34+ cells migrated poorly and did not contract their cytoskeleton upon treatment with the chemoattractant stromal cell-derived factor -1 (SDF-1), similar to the phenotype induced by PTX treatment. Gene expression studies comparing S1-transduced and control 32D cells uncovered four genes, expression of which was regulated by Gi protein blockade. Of interest, although SDF-1 signaling was inhibited, comparison between SDF-1-treated and untreated cells suggests that SDF-1 stimulation does not depend on de novo gene expression in these cells. Furthermore, when injected into nonobese diabetic/severe combined immunodeficient mice, seeding of S1-expressing 32D cells to bone marrow was largely blocked. CONCLUSION: Expression of S1 is an effective approach for studying long-term functional consequences of Gi protein blockade in hemopoietic cells in vitro and in vivo.

Base Sequence↗

PubMed2005

A capsid-modified helper-dependent adenovirus vector containing the beta-globin locus control region displays a nonrandom integration pattern and allows stable, erythroid-specific gene expression.

Gene therapy for hemoglobinopathies requires efficient gene transfer into hematopoietic stem cells and high-level erythroid-specific gene expression. Toward this goal, we constructed a helper-dependent adenovirus vector carrying the beta-globin locus control region (LCR) to drive green fluorescent protein (GFP) expression, whereby the LCR-GFP cassette is flanked by adeno-associated virus (AAV) inverted terminal repeats (Ad.LCR-beta-GFP). This vector possesses the adenovirus type 35 fiber knob that allows efficient infection of hematopoietic cells. Transduction and vector integration studies were performed in MO7e cells, a growth factor-dependent CD34(+) erythroleukemic cell line, and in cord blood-derived human CD34(+) cells. Stable transduction of MO7e cells with Ad.LCR-beta-GFP was more efficient and less subject to position effects and silencing than transduction with a vector that did not contain the beta-globin LCR. Analysis of integration sites indicated that Ad.LCR-beta-GFP integration in MO7e cells was not random but tethered to chromosome 11, specifically to the globin LCR. More than 10% of analyzed integration sites were within the chromosomal beta-globin LCR. None of the Ad.LCR-beta-GFP integrations occurred in exons. The integration pattern of a helper-dependent vector that contained X-chromosomal stuffer DNA was different from that of the beta-globin LCR-containing vector. Infection of primary CD34(+) cells with Ad.LCR-beta-GFP did not affect the clonogenic capacity of CD34(+) cells. Transduction of CD34(+) cells with Ad.LCR-beta-GFP resulted in vector integration and erythroid lineage-specific GFP expression.

Adenoviridae↗

PubMed2004

PTX-sensitive signals in bone marrow homing of fetal and adult hematopoietic progenitor cells.

Several examples suggest a relationship between in vitro migratory capacity and bone marrow (BM) homing. Pertussis toxin (PTX) is a potent inhibitor of serpentine receptor-associated inhibitory trimeric guanidine nucleotide binding (Gi) protein signals. As such, it blocks hematopoietic progenitor cell migration in vitro, but contrary to expectation, no effects on BM homing were observed in previous studies. We therefore re-examined the effect of PTX on homing of murine BM and fetal liver (FL). We found that BM homing of PTX-incubated progenitor cells (colony-forming cells in culture [CFU-Cs]) from BM or FL in irradiated and nonirradiated recipients was reduced by more than 75%, with a concomitant increase in circulating CFU-Cs in peripheral blood. Additional studies confirmed the functional significance of this reduction in homing: PTX-treated cells did not provide radioprotection, and their short-term engraftment in BM and spleen was drastically reduced. Furthermore, several approaches show that cell-intrinsic rather than host-derived mechanisms are responsible for the PTX-induced homing defect. In summary, we show that Gi protein signals are required for BM homing and, as such, provide a new example of the association between BM homing and in vitro migration. Moreover, our data suggest that the behavior of hematopoietic progenitors in obeying Gi signaling does not diverge from that of mature leukocytes.

Actins↗

PubMed2004

Transgenic Cre expression mice for generation of erythroid-specific gene alterations.

Transgenic mice that express Cre recombinase in erythroid cell lineages were developed so that genes affecting erythropoiesis/hematopoiesis may be altered without necessarily affecting fetus viability. A micro-LCR cassette-beta-globin promoter-Cre recombinase gene (microLCR-betapr-Cre) construct was synthesized and used to generate transgenic mice. Concurrently, we produced mice containing a microLCR-loxP-flanked beta sickle gene (microLCR-loxP-beta(S)-loxP) construct. microLCR-betapr-Cre mice with intact transgenes in variable copy number were identified. Cre expression was assessed by RNAse protection and RT-PCR. Cre function was ascertained by breeding to microLCR-loxP-beta(S)-loxP mice. We demonstrate that beta(S) expression was not detected in the blood of bigenics, but the gene was present in nonerythroid cells. Thus, excision of the loxP-flanked beta(S) gene was restricted to erythroid cell lineages.

Animals↗

PubMed2003

Current mechanistic scenarios in hematopoietic stem/progenitor cell mobilization.

Uncovering the molecular mechanisms governing the exit of stem/progenitor cells from bone marrow to peripheral blood at steady state or after their enforced migration has been an ongoing challenge. Recently, however, several new avenues or paradigms in mobilization have emerged from ever-expanding work in humans subjected to granulocyte colony-stimulating factor (G-CSF) mobilization, as well as from studies in normal and gene-deficient mouse models. Although these developments represent notable advances that met with considerable excitement, they have been quenched by surprising vacillations in subsequent research. This perspective highlights recent developments in mobilization along with their controversies. A full understanding of the directional cues that control the migratory behavior and the fate of stem/progenitor cells once they migrate out of bone marrow will await further experimentation, aiming to bridge our current gaps in knowledge.

Animals↗

PubMed2003

Megakaryocytes require thrombospondin-2 for normal platelet formation and function.

Mice that lack the matricellular angiogenesis inhibitor, thrombospondin-2 (TSP2), display a bleeding diathesis, despite normal blood coagulation and the lack of thrombocytopenia. Although platelets do not contain detectable levels of TSP2, TSP2-null platelets are compromised in their ability to aggregate in vivo in response to denudation of the carotid artery endothelium, and in vitro following exposure to adenosine diphosphate (ADP). Megakaryocytes (MKs) show high levels of TSP2 by immunohistochemical analysis of bone marrow. However, when cultured in vitro, MKs contain little TSP2 protein or mRNA. These findings suggest that most TSP2 is acquired from the bone marrow microenvironment. Consistent with this hypothesis, MKs take up recombinant TSP2 in an integrin-dependent manner when it is supplied in the culture medium. Furthermore, uptake of TSP2 in vitro affects MK differentiation and proplatelet formation. The functional significance of this process is supported by the presence of ultrastructural abnormalities in TSP2-null bone marrow, including extensive fragmentation of the peripheral zone in MKs and failure of this zone to form close associations with vascular sinuses. We conclude that the uptake of TSP2 by MKs from the marrow milieu is required for proper MK function and the release of functionally competent platelets.

Animals↗

PubMed2003

The role of G-protein signaling in hematopoietic stem/progenitor cell mobilization.

The directed migration of mature leukocytes to inflammatory sites and the lymphocyte trafficking in vivo are dependent on G protein-coupled receptors and delivered through pertussis toxin (Ptx)-sensitive Gi-protein signaling. In the present study, we explored the in vivo role of G-protein signaling on the redistribution or mobilization of hematopoietic stem/progenitor cells (HPCs). A single injection of Ptx in mice elicits a long-lasting leukocytosis and a progressive increase in circulating colony-forming unit-culture (CFU-C) and colony-forming unit spleen (CFU-S). We found that the prolonged effect is sustained by a continuous slow release of Ptx bound to red blood cells or other cells and is potentially enhanced by an indirect influence on cell proliferation. Plasma levels of certain cytokines (interleukin 6 [IL-6], granulocyte colony-stimulating factor [G-CSF]) increase days after Ptx treatment, but these are unlikely initiators of mobilization. In addition to normal mice, mice genetically deficient in monocyte chemotactic protein 1 (MCP-1), matrix metalloproteinase 9 (MMP-9), G-CSF receptor, beta2 integrins, or selectins responded to Ptx treatment, suggesting independence of Ptx-response from the expression of these molecules. Combined treatments of Ptx with anti-very late activation antigen (anti-VLA-4), uncovered potentially important insight in the interplay of chemokines/integrins, and the synergy of Ptx with G-CSF appeared to be dependent on MMP-9. As Ptx-mobilized kit+ cells display virtually no response to stromal-derived factor 1 (SDF-1) in vitro, our data suggest that disruption of CXCR4/SDF-1 signaling may be the underlying mechanism of Ptx-induced mobilization and indirectly reinforce the notion that active signaling through this pathway is required for continuous retention of cells within the bone marrow. Collectively, our data unveil a novel example of mobilization through pharmacologic modulation of signaling.

Animals↗

PubMed2003

Mutations in proto-oncogene GFI1 cause human neutropenia and target ELA2.

Mice lacking the transcriptional repressor oncoprotein Gfi1 are unexpectedly neutropenic. We therefore screened GFI1 as a candidate for association with neutropenia in affected individuals without mutations in ELA2 (encoding neutrophil elastase), the most common cause of severe congenital neutropenia (SCN; ref. 3). We found dominant negative zinc finger mutations that disable transcriptional repressor activity. The phenotype also includes immunodeficient lymphocytes and production of a circulating population of myeloid cells that appear immature. We show by chromatin immunoprecipitation, gel shift, reporter assays and elevated expression of ELA2 in vivo in neutropenic individuals that GFI1 represses ELA2, linking these two genes in a common pathway involved in myeloid differentiation.

Adult↗

PubMed2003

Bone marrow homing: the players, the playfield, and their evolving roles.

Although homing of hematopoietic cells to the bone marrow was described as a functional concept several decades ago, the analysis of its components and the molecular pathways involved remains an ongoing challenge. Because of the biologic and clinical significance of homing, a resurgence of studies delving into the mechanistic and molecular aspects of homing is appearing. These recent studies, highlighted in this brief review, emphasize newly appreciated roles of some known regulators and their interacting partners in homing, although some novel regulators also seem to enter the scene. The homing field still has disparities in experimental design and evaluation of data; however, the ever-expanding search for new information, together with the application of novel technologies, especially when combinations of approaches are used, will certainly bring our understanding of homing to a new level.

Animals↗

PubMed2003

Deletion of alpha4 integrins from adult hematopoietic cells reveals roles in homeostasis, regeneration, and homing.

We have explored the functional implications of inducible alpha4 integrin deletion during adult hematopoiesis by generating a conditional-knockout mouse model, and we show that alpha4 integrin-deficient hematopoietic progenitor cells accumulate in the peripheral blood soon after interferon-induced gene deletion. Although their numbers gradually stabilize at a lower level, progenitor cell influx into the circulation continues at above-normal levels for more than 50 weeks. Concomitantly, a progressive accumulation of progenitors occurs within the spleen. In addition, the regeneration of erythroid and myeloid progenitor cells is delayed during stress hematopoiesis induced by phenylhydrazine or by 5-fluorouracil, suggesting impairment in early progenitor expansion in the absence of alpha4 integrin. Moreover, in transplantation studies, homing of alpha4(-/-) cells to the bone marrow, but not to the spleen, is selectively impaired, and short-term engraftment is critically delayed in the early weeks after transplantation. Thus, conditional deletion of alpha4 integrin in adult mice is accompanied by a novel hematopoietic phenotype during both homeostasis and recovery from stress, a phenotype that is distinct from the ones previously described in alpha4 integrin-null chimeras and beta1 integrin-conditional knockouts.

Anemia, Hemolytic↗