Biomedical subjects

Georges Uzan

Publications and source records attributed to Georges Uzan.

15 recordsLinked to original sources

PubMed2006

Tripterine inhibits the expression of adhesion molecules in activated endothelial cells.

Cell adhesion molecules (CAM) expressed by vascular endothelium in response to cytokine stimulation play a key role in leukocyte adhesion to endothelium during the inflammatory response. Tripterine, a chemical compound of the Chinese plant Tripterygium wilfordii Hook f, displays anti-inflammatory properties in several animal models. However, mechanisms of its action are poorly understood. In the present study, we show that in inflammatory conditions, mimicked by tumor necrosis factor alpha (TNF-alpha) stimulation, pretreatment for 6 h with tripterine at nontoxic concentrations of 20-200 nM inhibits the expression of E-selectin, vascular cell adhesion molecule (CAM)-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) in human umbilical vein endothelial cells (HUVEC) in a dose-dependent manner. Tripterine (200 nM) almost completely inhibits expression of VCAM-1 [50% inhibitory concentration (IC50) = 52 nM] and ICAM-1 (IC50 = 51 nM) and 73% of E-selectin (IC50 = 94 nM). This inhibition effect is prominent, compared with that of dexamethasone, ibuprofen, methotrexate, or probucol, which revealed a much weaker inhibition at doses as high as 1 mM. Effects on endothelial CAM of other proinflammatory cytokines, such as interleukin-1beta and interferon-gamma, were also inhibited significantly by tripterine. Moreover, significant inhibition was equally observable in postincubation experiments. In addition, tripterine inhibited adhesion of human monocytes and T lymphocytes to TNF-alpha-stimulated HUVEC. Finally, tripterine inhibited TNF-alpha-driven CAM mRNA transcription and nuclear factor-kappaB nuclear (NF-kappaB) translocation. Hence, we describe a new mechanism of tripterine's anti-inflammatory action obtained at nanomolar concentrations, owing to the negative regulation of cytokine-induced adhesion molecule expression and adhesiveness in human endothelium.

Anti-Inflammatory Agents↗

PubMed2005

Low-molecular-weight fucoidan enhances the proangiogenic phenotype of endothelial progenitor cells.

Endothelial progenitor cell (EPC) transplantation is a potential means of inducing neovascularization in vivo. However, the number of circulating EPC is relatively small, it may thus be necessary to enhance their proangiogenic properties ex vivo prior to injection in vivo. Fucoidan has previously been shown to potentiate in vitro tube formation by mature endothelial cells in the presence of basic fibroblast growth factor (FGF-2). We therefore examined whether fucoidan, alone or combined with FGF-2, could increase EPC proangiogenic potency in vitro. EPC exposure to 10 microg/ml fucoidan induced a proangiogenic phenotype, including cell proliferation (p < 0.01) and migration (p < 0.01); moreover, differentiation into vascular cords occurred in the presence of FGF-2 (p < 0.01). This latter effect correlated with upregulation of the cell-surface #alpha6 integrin subunit of the laminin receptor (p < 0.05). Compared to untreated HUVEC, untreated EPC #alpha6 expression and adhesion to laminin were enhanced two-fold. Fucoidan treatment further enhanced HUVEC but not EPC adhesion to laminin. These results show that fucoidan enhances the proangiogenic properties of EPC and suggest that ex vivo fucoidan preconditioning of EPC might lead to increased neovascularization when injected into ischemic tissues.

Cell Proliferation↗

PubMed2005

Expression of Pitx2 in stromal cells is required for normal hematopoiesis.

Although the expression of Pitx2, a bicoid family homeodomain transcription factor, is highly regulated during hematopoiesis, its function during this process was not documented; we thus studied hematopoiesis in Pitx2-null mice. We found that Pitx2(-/-) embryos display hypoplastic livers with reduced numbers of hematopoietic cells, but these cells had normal hematopoietic potential, as evidenced by colony-forming assays, immature progenitor cell assays, and long-term repopulation assays. Because the microenvironment is also crucial to the development of normal hematopoiesis, we established Pitx2(-/-) and Pitx2(+/+) stromas from fetal liver and studied their hematopoietic supportive capacity. We showed that the frequency of cobblestone area-forming cells was 4-fold decreased when using Pitx2(-/-) stromal cells compared with Pitx2(+/+) stromal cells, whatever the Pitx2 genotype of hematopoietic cells tested in this assay. This defect was rescued by expression of Pitx2 into Pitx2(-/-) fetal liver stromal cells, demonstrating a major and direct role of Pitx2 in the hematopoietic supportive capacity of fetal liver stroma. Finally, we showed a reduced capacity of MS5 stromal cells expressing Pitx2 RNAi to support human hematopoiesis. Altogether these data showed that Pitx2 has major functions in the hematopoietic supportive capacity of fetal liver and adult bone marrow stromal cells.

Animals↗

PubMed2005

PAR-1 activation on human late endothelial progenitor cells enhances angiogenesis in vitro with upregulation of the SDF-1/CXCR4 system.

OBJECTIVE: The importance of PAR-1 in blood vessel development has been demonstrated in knockout mice. As endothelial progenitor cells (EPCs) are involved in postnatal vasculogenesis, we examined whether they express PAR-1 and whether stimulation by the peptide SFLLRN modulates their angiogenic properties. METHODS AND RESULTS: EPC expanded from human CD34+ cord blood cells expressed PAR-1. PAR-1 activation induced EPC proliferation in a concentration-dependent manner far more potently than that of human umbilical vein endothelial cells. PAR-1 activation also enhanced actin reorganization, promoting both spontaneous migration in a Boyden chamber assay and migration toward SDF-1 and VEGF. As shown by real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR), EPC stimulation by SFLLRN significantly enhanced the mRNA expression of SDF-1 and its receptor CXCR-4. PAR-1 activation also increased CXCR4 expression on EPC and induced SDF-1 secretion, leading to autocrine stimulation. PAR-1 stimulation by SFLLRN also increased the formation of capillary-like structures by EPC in Matrigel, and this effect was abrogated by anti-CXCR-4, anti-SDF-1, and MEK inhibitor pretreatment. CONCLUSIONS: Human EPCs express functional PAR-1. PAR-1 activation promotes cell proliferation and CXCR4-dependent migration and differentiation, leading to a proangiogenic effect.

Actin Cytoskeleton↗

PubMed2005

[Therapeutic potential of circulating endothelial cells].

The discovery of circulating endothelial progenitors (EPCs) in human blood has completely modified the concept of post natal angiogenesis. Many studies have further confirmed the existence of EPCs, their medullar origin and capacity to be integrated in vascular structures at the sites of neoangiogenesis. They suggest that EPCs could be naturally involved in the prevention of ischemia by participating directly to the formation of collateral vessels. These cells have a high tropism for the sites of angiogenesis. They may thus be used as a powerful tool to prevent or treat ischemic diseases and constitute an alternative to the risky local injection of pro-angiogenic growth factors. The efficiency of bone marrow cells autologous transplantation was recently proved by the first clinical trials. Bone marrow mononuclear cells locally injected to patients with limb or cardiac ischemia have been shown to improve reperfusion in ischemic tissues. These trials are still preliminary: they were performed with heterogeneous cells only on few patients and were not randomized. However they raise important questions, essentially on the necessity of cell purification prior to injection and on the nature of the bone marrow cells which are really involved in ischemic tissue regeneration. Further investigations are then required to improve the cell therapy outcome by 1) using more defined cell population, 2) clearly demonstrating the long term improvement of vascular function and 3) performing extensive analysis of the possible side effects.

Bone Marrow Cells↗

PubMed2005

Presence of endothelial progenitor cells, distinct from mature endothelial cells, within human CD146+ blood cells.

CD146 is an adhesion molecule present on endothelial cells throughout the vascular tree. CD146 is also expressed by circulating endothelial cells (CECs) widely considered to be mature endothelial cells detached from injured vessels. The discovery of circulating endothelial progenitor cells (EPCs) originating from bone marrow prompted us to investigate whether CD146 circulating cells could also contains EPCs. We tested this hypothesis using an approach combining elimination of CECs by an adhesion step, followed by immunomagnetic sorting of remaining CD146+ cells from the non adherent fraction of cord blood mononuclear cells. When cultured under endothelial-promoting conditions, these cells differentiated as late outgrowth endothelial colonies: they grew as a cobblestone monolayer, were uniformly positive for endothelial markers and did not express leukocyte antigens. They highly proliferated and were expanded in long-term culture without alterations of their phenotypic and functional properties (Dil-ac-LDL uptake, wound repair, capillary-like network formation, and TNFalpha response). Moreover, these cells colonized a Matrigel plug in immunodeficient mice (NOD/SCID). Finally, using 4-color flow cytometry analysis of purified CD34+ cells, we clearly discriminated, CD146+ EPCs (CD146+ CD34+ CD45+ CD133+ or CD117+), and CD146+ CECs (CD146+ CD34+, CD45- CD133- or CD117-), both in cord and adult peripheral blood.The relative proportions of the two CD146+ subsets varied in patients with myocardial infarction as compared to healthy subjects. Our study establishes that, beside CECs, CD146+ circulating cells contain a subpopulation of EPCs with potential use in proangiogenic therapy. In addition, the dual measurement of CD146+ CECs and CD146+ EPCs offers a promising tool for monitoring vascular injury/regeneration processes in clinical situations.

Animals↗

PubMed2004

[Therapeutic use of stem cells. II. Adult stem cells].

Many degenerative diseases are not curable by means of classical medicine. The long term objective of cell therapy is to treat the patients with their own stem cells that could be either purified from the diseased organ or from "reservoirs" of stem cells such as that constituted by the bone marrow. The existence of stem cells in the organs or reservoirs is now established in vitro and in some cases, in animal models. Numbers of technical problems linked to the scarcity of these cells still delay the clinical use of purified stem cells. However, clinical protocols using heterogeneous cell populations have already started to treat a growing number of diseases. In some case, autologous cells can be used, as it is the case for bone marrow transplantation in blood diseases. Mesenchymal cells, also purified from the bone marrow are currently used in orthopaedic diseases. Because these cells reveal a broad differentiation potential, active research programs explore their possible use for treatment of other diseases. Bone marrow also contains vascular stem cells that could be active in reappearing defective vessels responsible for ischaemic diseases. Indeed, clinical trials in which bone marrow cells are injected in the cardiac muscle of patients with myocardial infarction or in the leg muscle (gastrocnemius) of patients with hind limb ischaemia have already started. Artificial skin prepared from skin biopsies is used for the reconstitution of the derma of severely burned patients. Clinical trials have also started, using allogenic cells. The patients must be treated by immunosuppressive drugs. Neurodegenerative diseases such as Parkinson have been successfully treated by intra-cerebral injection of foetal neurones. Pancreatic islets implanted in the liver have shown to re-establish a normal glycaemia in diabetic patients. However, all these clinical trials use differentiated cells or at least progenitors which display differentiation potential and lifetime much more restricted than those of stem cell. Numbers of laboratories are currently working to improve stem cell purification and expansion. This is a prerequisite to use these stem cells as a more efficient second generation cell therapy product.

Adult↗

PubMed2004

[Therapeutic use of stem cells].

Stem cells display important capacities of self renewing, proliferation and differentiation. Because those present in the embryo have the more remarkable properties, their potential use in the therapy of until now incurable degenerative diseases have been envisioned. Embryonic stem (ES) cells are located in the inner mass of the balstocyst at early stages of the development. Even in long-term cultures they still retain their undifferentiated features. Under specific culture conditions, ES cells can be committed into a variety of differentiation pathways, giving rise to large amounts of cells corresponding to different tissues (neurones, cardiomyocytes, skeletal muscle, etc.). However, producing these tissues from already established ES cell lines would lead to immune rejection when transplanted to patients. To prevent this pitfall and using the expertise accumulated by animal cloning by nucleus transfer, it has been proposed to adapt this technique to human ES cells. The therapeutic cloning consists in transferring the nucleus of somatic stem cells isolated from the patient into an enucleated oocyte, to allow blastocyst development from which ES cells will be derived. From these stem cells, compatible tissues will be then produced. The problem is that it is in theoretically possible to reimplant the cloned blastocyst into a surrogate mother for obtaining a baby genetically identical to the donor. This is called reproductive cloning. This worrying risk raises important ethic and legal questions.

Cell Differentiation↗

PubMed2004

Biosynthesis of FVIII in megakaryocytic cells: improved production and biochemical characterization.

Haemophilia A is an attractive target for gene therapy. We designed a haemophilia A gene therapy strategy involving the genetic modification of haematopoietic stem cells to achieve tissue-specific expression of a factor VIII (FVIII) transgene in the megakaryocytic lineage. Platelets would then serve as vehicles to store the expressed FVIII and deliver the coagulation factor at the site of vascular injury. A local correction of the haemostasis defect could, therefore, be expected following platelet activation and secretion. In this study, we demonstrated that a model of haematopoietic cell lines (Dami cells) could produce a correctly processed FVIII. FVIII transgenes were placed under the control of the human platelet glycoprotein IIb (GPIIb) promoter and used for stable transfection of the Dami megakaryocytic cell line. The highest FVIII production was obtained when the FVIII transgene contained a factor IX intron 1 gene sequence inserted in the FVIII intron 1 and 13 sites. Reverse transcription polymerase chain reaction demonstrated that the splicing of these introns was complete. Recombinant FVIII (rFVIII) produced in Dami cells was a biologically active molecule (specific activity: 5664 IU/mg) that was correctly glycosylated and sulphated. This recombinant FVIII protein exhibited biochemical characteristics after deglycosylation or thrombin activation that were comparable to a commercially available B-domainless rFVIII. These results demonstrate the advantages of a modified FVIII transgene and represent the first biochemical characterization of megakaryocyte-produced FVIII.

Bioreactors↗

PubMed2003

Human endothelial cells derived from circulating progenitors display specific functional properties compared with mature vessel wall endothelial cells.

Endothelial progenitor cells (EPCs) were shown to be present in systemic circulation and cord blood. We investigated whether EPCs display specific properties compared with mature endothelial cells. Human cord blood CD34+ cells were isolated and adherent cells were amplified under endothelial conditions. Expression of specific markers identified them as endothelial cells, also called endothelial progenitor-derived cells (EPDCs). When compared to mature endothelial cells, human umbilical vein endothelial cells (HUVECs) and human bone marrow endothelial cells (HBMECs), endothelial markers, were expressed to the same extent except for KDR, which is expressed more in EPDCs. They display a higher proliferation potential. Functional studies demonstrated that EPDCs were more sensitive to angiogenic factors, which afford these cells greater protection against cell death compared with HUVECs. Moreover, EPDCs exhibit more hematopoietic supportive activity than HUVECs. Finally, studies in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice demonstrated that human circulating EPCs are able to colonize a Matrigel plug. EPDCs display the morphology and phenotype of endothelial cells. Their functional features indicate, however, that although these cells have undergone some differentiation steps, they still have the properties of immature cells, suggesting greater tissue repair capabilities. Future use of in vitro amplified peripheral blood EPDCs may constitute a challenging strategy for cell therapy.

Animals↗

PubMed2003

Molecular mechanism of the anti-cancer activity of cerivastatin, an inhibitor of HMG-CoA reductase, on aggressive human breast cancer cells.

Statins are currently used for the treatment of hypercholesterolemia. Recently, we demonstrated that cerivastatin also reduces the proliferation and invasion of aggressive breast cancer cells, MDA-MB-231. In this report, a molecular mechanism to explain its anti-cancer action is proposed by combining the study of cerivastatin effect on both gene expression (microarray) and signal transduction pathways. Firstly, the expression of 13 genes was modified by cerivastatin and confirmed at protein level. They could contribute to the inhibition of both cell proliferation (down-regulation of cyclin D1, PCNA, c-myc and up-regulation p21(Waf1), p19(INK4d), integrin beta8) and cell invasion, either directly (decrease in u-PA, MMP-9, u-PAR, PAI-1 and increase in anti-oncogenes Wnt-5a and H-cadherin) or indirectly by stimulating an anti-angiogenic gene (thrombospondin-2). The anti-angiogenic activity was confirmed by in vivo experiments. Secondly, we demonstrated that the biochemical mechanism of its anti-cancer action could be mainly explained by the inhibition of RhoA-dependent cell signalling. This hypothesis was supported by the fact that a RhoA inhibitor (C3 exoenzyme) or a dominant negative mutant RhoA (N19RhoA) induced similar effects to those of cerivastatin. In conclusion, cerivastatin, by preventing RhoA prenylation, inhibits (i) the RhoA/ROCK pathway, leading to defective actin stress fibres formation responsible for the loss of traction forces required for cell motility and (ii) the RhoA/FAK/AKT signalling pathway that could explain the majority of cancer-related gene modifications described above. Thus, the inhibition of RhoA cell signalling could be a good strategy in therapy of aggressive forms of breast cancer.

ADP Ribose Transferases↗

PubMed2003

Insights in the molecular mechanisms of the anti-angiogenic effect of an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) reduce the risk of coronary event by cholesterollowering dependent and independent mechanisms. We have already described that the inhibitory effect of cerivastatin on angiogenesis contribute to the cholesterol-independent beneficial effect and was due to the inhibition of the cell signaling cascade RhoA/FAK/Akt. In this study, new insights in the molecular mechanism of action were provided. It indicates an inhibition of exposure of alpha V beta 3 integrin on cell membrane and a modification of gene expression. The inhibition of angiogenesis could be related to 1) an increase in genes involved in the inhibition of cell proliferation (p19(INK4), p21(Waf/Cip1),Wnt-5a), the inhibition of cell migration (Rho-GDI 1, alpha E-catenin) and 2) a downregulation of genes involved in angiogenesis (PAI-1, Vitronectin, HoxD3, Notch4) or in cell invasion (Semaphorin E). In addition, DNA repair protein genes (MLH1, XRCC1) were increased. This study may indicate new biological interest of genes involved in angiogenesis control.

Angiogenesis Inhibitors↗

PubMed2002

Fetal liver stroma consists of cells in epithelial-to-mesenchymal transition.

Liver becomes the predominant site of hematopoiesis by 11.5 dpc (days after coitus) in the mouse and 15 gestational weeks in humans and stays so until the end of gestation. The reason the liver is the major hematopoietic site during fetal life is not clear. In this work, we tried to define which of the fetal liver microenvironmental cell populations would be associated with the development of hematopoiesis and found that a population of cells with mixed endodermal and mesodermal features corresponded to hematopoietic-supportive fetal liver stroma. Stromal cells generated from primary cultures or stromal lines from mouse or human fetal liver in the hematopoietic florid phase expressed both mesenchymal markers (vimentin, osteopontin, collagen I, alpha smooth muscle actin, thrombospondin-1, EDa fibronectin, calponin, Stro-1 antigens, myocyte-enhancer factor 2C) and epithelial (alpha-fetoprotein, cytokeratins 8 and 18, albumin, E-cadherin, hepatocyte nuclear factor 3 alpha) markers. Such a cell population fits with the description of cells in epithelial-to-mesenchymal transition (EMT), often observed during development, including that of the liver. The hematopoietic supportive capacity of EMT cells was lost after hepatocytic maturation, induced by oncostatin M in the cell line AFT024. EMT cells were observed in the fetal liver microenvironment during the hematopoietic phase but not in nonhematopoietic liver by the end of gestation and in the adult. EMT cells represent a novel stromal cell type that may be generated from hepatic endodermal or mesenchymal stem cells or even from circulating hematopoietic stem cells (HSCs) seeding the liver rudiment.

Animals↗

PubMed2002

The cytoplasmic domain of Mpl receptor transduces exclusive signals in embryonic and fetal hematopoietic cells.

The Mpl receptor plays an important role at the level of adult hematopoietic stem cells, but little is known of its function in embryonic and fetal hematopoiesis. We investigated the signals sent by the MPL cytoplasmic domain in fetal liver hematopoietic progenitors and during embryonic stem (ES) cell hematopoietic commitment. Mpl was found to be expressed only from day 6 of ES cell differentiation into embryoid bodies. Therefore, we expressed Mpl in undifferentiated ES cells or in fetal progenitors and studied the effects on hematopoietic differentiation. To avoid the inadvertent effect of thrombopoietin, we used a chimeric receptor, PM-R, composed of the extracellular domain of the prolactin receptor (PRL-R) and the transmembrane and cytoplasmic domains of Mpl. This allowed activation of the receptor with a hormone that is not involved in hematopoietic differentiation and assessment of the specificity of responses to Mpl by comparing PM-R with another PRL-R chimeric receptor that includes the cytoplasmic domain of the erythropoietin receptor (EPO-R) ([PE-R]). We have shown that the cytoplasmic domain of the Mpl receptor transduces exclusive signals in fetal liver hematopoietic progenitors as compared with that of EPO-R and that it promotes hematopoietic commitment of ES cells. Our findings demonstrate for the first time the specific role of Mpl in early embryonic or fetal hematopoietic progenitors and stem cells.

Animals↗

PubMed2002

Forced expression of p21 in GPIIb-p21 transgenic mice induces abnormalities in the proliferation of erythroid and megakaryocyte progenitors and primitive hematopoietic cells.

OBJECTIVE: p21(WAF1/Cip/kip) and p27(Kip1) are cyclin-dependant kinase inhibitors controlling cell-cycle exit and differentiation of numerous cell types. Among hematopoietic cells, megakaryocytes express high levels of p21, while in erythroid cells, p27(Kip1) is predominant. As p21 and p27 could display overlapping functions and as megakaryocytes and erythroid cells derive from a bipotent progenitor, we developed an in vivo model to determine the specific role of p21 in controlling the proliferation/differentiation balance of erythroid and megakaryocytic progenitors. METHODS: Transgenic mice that overexpressed p21 under the control of the human GPIIb promoter in early progenitors and along megakaryocytic differentiation were generated. Different subsets of hematopoietic progenitors (BFU and CFU) and primitive cells (CAFC, LTC-IC) were analyzed by methylcellulose assay. Phenotypic evolution and clonogenic properties of the lin(-) population were analyzed along erythroid and megakaryocytic differentiation. RESULTS: We observed p21 ectopic expression in early hematopoietic progenitors (lin(-)Sca(+)), megakaryocytes, and, to a lesser extent, erythroid cells. This expression induced an important decrease in the number of CFU-MK, BFU-E, CFU-E, primitive progenitors (CAFC day 35), and LTC-IC, but did not affect the maturation process of these cells and the blood cell count. CONCLUSIONS: We show that variation of p21 expression level changes the fate of hematopoietic cells by favoring either proliferation or differentiation pathways. This effect of p21 is exerted not only at the level of primitive progenitors but also in more mature progenitors. However, in vivo, a systemic compensation mechanism is most likely activated in response to variations of the flow of progenitor production.

Animals↗