Characterization of the Procoagulant Potential of Endothelial Colonies Forming Cells and Comparison with Two Cell Therapy Products: Relevance for Vascular Cell Therapy

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5317-5317
Author(s):  
Wendy Cuccuini ◽  
Claire Tournois ◽  
Gael Poitevin ◽  
Bernard Pignon ◽  
Marie Antoinette Sevestre ◽  
...  
Keyword(s):  
Cell Therapy ◽  
Cord Blood ◽  
Tissue Factor ◽  
Thrombin Generation ◽  
Mononuclear Cells ◽  
Procoagulant Activity ◽  
Inflammatory Conditions ◽  
Cell Based Therapy ◽  
Cd34 Cells

Abstract Abstract 5317 Background: Endothelial colonies forming cells (ECFC) are good candidates for cell-based therapy in cardiovascular diseases. Concerns have been raised about the potential risks of ECFC-based cell therapy, in terms of thrombogenicity, particularly in inflammatory conditions. We published that cord blood ECFC (cb-ECFC) express Tissue Factor (TF) in response to TNFα. TF/FVIIa interaction did not modify in vitro the non-procoagulant properties of TNFα stimulated cb-ECFC [Cuccuini et al, JTH 2010]. We also reported, in a monocyte model, a co-expression of TF and metalloproteinase MMP-9, with possible consequences in terms of haemostasis and angiogenesis [Poitevin et al, JTH 2008]. Aim of the study: The objective of this study was: 1- to further evaluate the procoagulant potential of ECFC and the capacity to exhibit MMP activity; 2- to evaluate whether TF and MMP could be detected in two different cell therapy products (CTP). Methods: ECFC were obtained in culture after isolation of CD34+ cells from cord blood (as previously described). CTP were obtained from an ongoing clinical trial evaluating cell therapy to improve vascularization in chronic critical limb ischemia (NIH NCT00533104). Two different CTP were analyzed: 1- Peripheral blood mononuclear cells (PB-MNC) obtained by cytapheresis without any mobilization (n= 11); 2- Bone marrow mononuclear cells (BM-MNC) (n= 11). CTP are characterized in flow cytometry, in terms of cell composition, including CD34+ cells; CD34+/CD133+/VEGF-R2+ cells were considered as endothelial progenitor cells. Results: cb-ECFC can express procoagulant TF in response to TNFa (10 ng/mL). The expression of TF was compared with purified monocytes, CD34+ cells, mature endothelial cells (HUVEC), PB-MNC and BM-MNC. TF mRNA were detected in CD34+ cells. As microparticles (MPs) are strongly procoagulant, we evidenced that cb-ECFC produced CD146+/Annexin V+/TF+ MPs in response to TNFa. These MPs can trigger thrombin generation. The analysis of conditioned supernatants of cb-ECFC in response to TNFa indicated that the majority of TF is borne by MP, but a small amount of soluble TF is present, in the absence of MP. This soluble TF does not induce thrombin generation. Using Q-PCR, we showed that cb-ECFC expressed Tissue Factor Pathway Inhibitor (TFPI). This expression of TFPI mRNA was not increased by TNFa. The analysis of TNFa-conditioned cb-ECFC supernatants showed that the amount of TFPI protein is a 20 fold lower than plasmatic levels of free-TFPI. We next examined the presence of TF mRNA in both CTP. TF mRNA were estimated to a 2 fold higher in BM-MNC than in cb-ECFC (baseline). On the contrary, TF mRNA were a 5 fold lower in PB-MNC than in cb-ECFC. In both CTP, the alternatively spliced form (as-TF) represented a very minor part of TF. The percentage of CD14+ cells was respectively 17 % and 26 % in BM-MNC and in PB-MNC. The amount of TF mRNA in PB-MNC was not statistically different from unstimulated purified monocytes. On the contrary, TF mRNA were a 7 fold higher in BM-MNC in comparison with PB-MNC or monocytes. The percentage of CD34+ cells was low: 2.6 % in BM-MNC and 0.1 % in PB-MNC. TF mRNA were higher in CD34+ cells than in BM-MNC (3 fold) and in PB-MNC (27 fold). As we previously reported a co-regulation of TF with MMP, we first analyzed the expression of MMP-2 and MMP-9 in cb-ECFC. Using Q-PCR, we found that cb-ECFC strongly expressed MMP-2 whereas the expression of MMP-9 was very weak. This profile was comparable in cb-ECFC and HUVEC. Using zymography, we confirmed the expression of MMP-2 but not MMP-9, in response to TNFa. We next analyzed CTP. In BM-MNC, MMP-2 and MMP-9 were expressed at a very high level. In PB-MNC, MMP-2 and MMP-9 could be detected but at a very low level. Conclusion: Taken together, our results show that ECFC, BM-MNC and PB-MNC can express TF. The procoagulant activity of ECFC is also the consequence of MP formation in inflammatory conditions. The profile of MMP in CTP remains to be clarified in terms of cell origin and possible influence on thrombotic and angiogenic properties. In our hands, the inhibition of TF can limit procoagulant activity with no effect on proangiogenic properties in vitro. In terms of clinical relevance, one single patient treated with PB-MNC, developed a deep vein thrombosis at the site of injection. It is therefore mandatory to evaluate the procoagulant potential of CTP in the context of vascular repair. Disclosures: No relevant conflicts of interest to declare.

Abstract 14209: A New in vitro Methodology to Evaluate Blood Mononuclear Cells Regenerative Capacity in Diabetes Patients With Acute Myocardial Infarction

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Amankeldi A Salybekov ◽  
Katsuaki Sakai ◽  
Makoto Natsumeda ◽  
Kosit Vorateera ◽  
Yuji Ikari ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Acute Myocardial Infarction ◽  
Cell Therapy ◽  
Mononuclear Cells ◽  
Roc Curve Analysis ◽  
Regenerative Capacity ◽  
Cd34 Cells ◽  
Blood Mononuclear Cells ◽  
M1 Type

Introduction & Hypothesis: Diabetes mellitus patients’(DMP) peripheral blood mononuclear cells (PBMNC) regenerative capacity level is impaired. An in vitro evaluation of PBMNC pre/post vasculogenic conditioning (VC) facilitates the assessment of immune cells regenerative potential (H1) and possible cell therapy for DMP with acute myocardial infarction (AMI) (H2). Materials & Methods: Eighteen DMP with the diagnosis of AMI enrolled. Blood drawn in heparin-coated syringes from AMI patients (between day 3 to 7) along with sixteen healthy control. Isolated PBMNC regenerative capability evaluated pre and post VC ( Fig 1 ) with EPCs colony formation assay/unit (EPC-CFA/U) and flow cytometry analysis. Results: An in vitro EPC-CFA revealed that DMP fresh PBMNC derived definitive EPC (DEPC) decreased compared to control. The differentiation rate of EPC, definitive vs. primitive in control groups composed equal (50%, PEPC vs. 50%, DEPC) while in DMP, PEPC prevails (70% vs. 30%). After VC, DEPC-CFU markedly increased while PEPC-CFU decreased, indicating EPC qualitatively and quantitatively improvement in DMP (Control, PBMNC vs. VC P>0.001; DMP, PBMNC vs. VC, P>0.01). DMP glycoalbumin and Hb1Ac inversely correlated with CD34+ cells (r= -0.48, P>0.03) while VC recovered CD34+ cells (r= 0.17, P<0.54). ROC curve analysis also confirmed that the CD34+ cell number is an independent risk classifier of cardiac vessel lesion (AUC=0.85, P>0.002). In contrast, VC preserved from the senescence by expansion and differentiation of CD34+ (AUC=0.54, P<0.7). Proinflammatory M1 type significantly increased in DMP compared to Control (P>0.03), while VC shifted the M1 type phenotype toward M2 type (P>0001). Conclusion: Our EPC-CFA enables us to precisely assess impaired EPC function, while VC enhanced differentiation from PEPC toward DEPC. Furthermore, these methodologies facilitate the evaluation of RACs capacities such as EPC, M1/M2, and Treg cells in DMP with AMI for cell therapy.

scholarly journals Increased in vitro and in vivo generation of procoagulant activity (tissue factor) by mononuclear phagocytes after intralipid infusion in rabbits

Blood ◽  
1985 ◽  
Vol 65 (6) ◽  
pp. 1391-1395 ◽  
Author(s):  
P Montemurro ◽  
A Lattanzio ◽  
G Chetta ◽  
L Lupo ◽  
L Caputi-Iambrenghi ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Mononuclear Cells ◽  
Mononuclear Phagocyte ◽  
Mononuclear Phagocytes ◽  
Procoagulant Activity ◽  
Sterile Saline ◽  
Functional Changes ◽  
Before And After

Abstract Intralipid, a fat emulsion widely used in parenteral nutrition, can produce marked functional changes of the mononuclear phagocyte system. We investigated the effect of Intralipid administration on the generation of procoagulant activity by rabbit mononuclear phagocytes. Two groups of ten rabbits given either a single infusion of Intralipid 10% or a similar volume of sterile saline were studied before and after infusion. Procoagulant activity was measured on isolated blood mononuclear cells after incubation with and without endotoxin, using a one-stage clotting assay. Cells from animals infused with Intralipid produced significantly more procoagulant activity than controls (P less than .01). Results were similar when freshly collected whole blood was incubated with and without endotoxin, and procoagulant activity was measured on subsequently isolated mononuclear cells (P less than .01). In addition, when rabbits were given a single injection of endotoxin, blood and spleen mononuclear cells harvested 50 to 60 minutes after the injection from animals pretreated with Intralipid expressed five to seven times more procoagulant activity than did cells from animals pretreated with saline. In all instances, procoagulant activity was identified as tissue factor. These findings suggest that Intralipid may cause functional changes in mononuclear phagocytes, resulting in increased production of tissue factor on incubation in short-term culture in vitro and in response to endotoxin in vivo.

Impact of V617F JAK2 Mutation on Monocyte Tissue Factor and Procoagulant Activity in Patients with Essential Thrombocythemia(ET) or Polycythemia VERA (PV)

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3736-3736
Author(s):  
Anna Falanga ◽  
Alfonso Vignoli ◽  
Marina Marchetti ◽  
Laura Russo ◽  
Marina Panova-Noeva ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Blood Coagulation ◽  
Thrombin Generation ◽  
Procoagulant Activity ◽  
Jak2v617f Mutation ◽  
Wild Type ◽  
Coagulation Activation ◽  
Allele Burden ◽  
Generation Capacity

Abstract Clinical data suggest an increased thrombotic risk in patients with ET or PV carrying the JAK2V617F mutation. Laboratory data from our group show that ET patients carrying the JAK2V617F mutation are characterized by an enhanced platelet and neutrophil activation status (Falanga et al, Exp Hem 2007) and blood coagulation activation (Marchetti et al, Blood 2008), as compared to JAK2 wild-type ET. Since monocytes significantly contribute to blood coagulation activation as an important source of circulating tissue factor (TF), in this study we aimed to characterize the prothrombotic phenotype of monocytes from ET and PV patients and to evaluate whether and to what extent it is influenced by the JAK2V617F mutation. Twenty-four ET patients (10 JAK2 wild-type; 14 JAK2V617F carriers with 2%–35% mutant allele burden), 27 PV patients (all JAK2V617F carriers, 16 with 9%– 44% and 11 with 60%–100% allele burden, respectively), and 20 age-matched healthy subjects (controls, C) were enrolled into the study. Monocyte-associated TF antigen was measured on the cell surface by whole blood flow-cytometry, in both basal condition and after in vitro stimulation by bacterial endotoxin (lypopolysaccharide, LPS), as well as in cell lysates by ELISA. Monocyte procoagulant activity was evaluated by the Calibrated Automated Thrombogram (CAT) as the capacity of isolated monocyte lysates to induce thrombin generation in normal pool plasma. In basal conditions, significantly (p<0.05) higher surface levels of TF were measured on monocytes from ET (17.1±3.2% positive cells) and PV (24.4±3.7% positive cells) patients compared to C (8.2±1.9% positive cells). Similarly, the total TF antigen content of cell lysates was significantly increased in patients compared to C. The analysis of the data according to JAK2V617F mutational status, showed a gradient of increased TF expression starting from JAK2V617F negative patients (11.7±2.5%), versus JAK2V617F ET and PV subjects with <50% allele burden (20.3±3.6% and 23.2±2.8%, respectively), versus JAK2V617F PV patients with >50% allele burden (26.1±4.2%). The in vitro LPS stimulation significantly increased TF expression on monocytes from all study subjects and C compared to non-stimulated monocytes (p<0.05 for all groups), with a more elevated expression by monocytes from PV and ET patients compared to C. However, the relative increase in TF expression was greater in C (=3.7 fold) compared to both ET (=2.2 fold) and PV (=2 fold) patients. As observed in basal conditions, LPS-induced TF was higher in JAK2V617F positive patients as compared to negative, with the highest expression in JAK2V617F PV carriers with >50% allele load. Thrombin generation induced by monocytes from ET and PV patients was significantly increased compared to controls, as determined by significantly higher thrombin peaks (ET=145±12, PV=142±17, C=72.2±5 nM), and quantity of thrombin generated in time, i.e. the endogenous thrombin potential (ETP) (ET=1143±34, PV=1074±64, C=787±58 nM*min). The JAK2V617F PV subjects with >50% allele burden presented with the highest thrombin generation capacity (peak= 184±34 nM; ETP= 1268±32 nM). Our data indicate that the expression of the JAK2V617F mutation in ET and PV patients may confer to monocytes a different hemostatic phenotype in terms of increased expression of surface TF and thrombin generation capacity. These findings are in agreement with the previous observation of a hypercoagulable state associated with this mutation and suggest a new mechanism linking hemostatic cellular phenotypic alteration to genetic dysfunction in patients with myeloproliferative disease.

Characterization of Angiogenic Potential of Late Endothelial Progenitor Cells on Human Cord Blood and Bone Marrow

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5404-5404
Author(s):  
Eun-Sun Yoo ◽  
Jee-Young Ahn ◽  
KiHwan Kwon ◽  
Soo-Ah Oh ◽  
Moon-Young Choi ◽  
...  
Keyword(s):  
Cord Blood ◽  
Progenitor Cells ◽  
Endothelial Progenitor Cells ◽  
Adhesion Molecule ◽  
Mononuclear Cells ◽  
Marker Genes ◽  
Endothelial Progenitor ◽  
Angiogenic Potential ◽  
Cell Based Therapy

Abstract Background: The identification of circulating endothelial progenitor cells (EPCs) has revolutionized approaches to cell-based therapy for injured and ischemic tissues. Recently, we have demonstrated that there are 2 distinct types of EPCs from UCB having different biologic properties for angiogenic capabilities in vitro and in vivo. In present study, the aim is to directly compare umbilical cord blood (UCB)- and BM-derived late EPC surface phenotypes and in vitro functional capacity. Methods: Mononuclear cells from UCB and BM cultured using EGM-2 medium with VEGF, IGF-1 and FGF for 21 days. Late outgrowing endothelail cells(late OECs) which were in peak growth at third weeks of culture were analyzed for expression of various surface markers by flow cytometry/RT-PCR/IF, tube formation in Matrigel plates, proliferation assay, endothelial colony assay and the role of SDF-1/VEGF on migration. Results: The adherent cells after culture of 7 days exhibited a fibroblast like shape in BM and a cobblestone shaped cells in UCB. Although two sources of OECs were comparable in expression of endothelial and various adhesion molecule markers, BM-derived OECs contained higher proportion of cells expressing smooth muscle cell markers(SMMHC), several adhesion molecule(CD49d, CD62L and VCAM-1), whereas the expression of CXCR-4, PECAM-1 and CD62E and expression of mRNA on endothelial marker genes were higher in UCB-derived OECs. UCB-OECS stained positive for uptake of acetylated low-density lipoprotein and had more migratory ability in the presence of SDF-1 and VEGF compared with BM-OECs. Both sources OECs effectively formed capillary tubes in Matrigel plates. Conclusion: We directly compared OECs derived from UCB and BM and two source of OECs differ in aspect of several adhesion molecule and angiogenic potential in vitro. These difference of UCB render it potentially advantageous for human therapeutic OECs applications for potential applications for a “cell therapy” in the situations on vascular injuries when compared with patients-derived BM.

scholarly journals Direct growth suppression of myeloid bone marrow progenitor cells but not cord blood progenitors by human cytomegalovirus in vitro

Blood ◽  
1996 ◽  
Vol 88 (7) ◽  
pp. 2510-2516 ◽  
Author(s):  
M Holberg-Petersen ◽  
H Rollag ◽  
S Beck ◽  
I Overli ◽  
G Tjonnfjord ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cord Blood ◽  
Clinical Isolate ◽  
Human Cytomegalovirus ◽  
Mononuclear Cells ◽  
Colony Growth ◽  
Structural Protein ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Recently, considerable interest has arisen as to use cord blood (CB) as a source of hematopoietic stem cells for allogenic transplantation when bone marrow (BM) from a familial HLA-matched donor is not available. Because human cytomegalovirus (HCMV) has been shown to inhibit the proliferation of BM progenitors in vitro, it was important to examine whether similar effect could be observed in HCMV-infected CB cells. Therefore, the effect of HCMV challenge on the proliferation of myeloid progenitors from BM and CB was compared using both mononuclear cells (MNC) and purified CD34+ cells. A clinical isolate of HCMV inhibited the colony formation of myeloid BM progenitors responsive to granulocyte-macrophage colony-stimulating factor (CSF), granulocyte-CSF, macrophage-CSF, interleukin-3 (IL-3) and the combination of IL-3 and stem cell factor (SCF). In contrast, colony growth of CB progenitors was not affected. In addition, HCMV inhibited directly the growth of purified BM CD34+ cells responsive to IL-3 and SCF in single cell assay by 40%, wheras the growth of CD34+ progenitors obtained from CB was not suppressed. The HCMV lower matrix structural protein pp65 and HCMV DNA were detected in both CB and BM CD34+ cells after in vitro challenge. However, neither immediate early (IE)-mRNA nor IE proteins were observed in infected cells. Cell cyclus examination of BM and CB CD34+ cells revealed that 25.7% of BM progenitors were in S + G2/ M phase wheras only 10.7% of the CB progenitors. Thus, a clinical isolate of HCMV directly inhibited the proliferation of myeloid BM progenitors in vitro wheras CB progenitors were not affected. This difference in the susceptibility of CB and BM cells to HCMV may partly be caused by the slow cycling rate of naive CB progenitors compared to BM progenitors at the time of infection.

scholarly journals Mesenchymal Stem Cell-Derived Microvesicles Support Ex Vivo Expansion of Cord Blood-Derived CD34+Cells

2016 ◽  
Vol 2016 ◽  
pp. 1-13 ◽  
Author(s):  
Hui Xie ◽  
Li Sun ◽  
Liming Zhang ◽  
Teng Liu ◽  
Li Chen ◽  
...  
Keyword(s):  
Cord Blood ◽  
Progenitor Cells ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Hematopoietic Stem ◽  
Promising Therapeutic Approach ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Mesenchymal stem cells (MSCs) are known to support the characteristic properties of hematopoietic stem and progenitor cells (HSPCs) in the bone marrow hematopoietic microenvironment. MSCs are used in coculture systems as a feeder layer for the ex vivo expansion of umbilical cord blood (CB) to increase the relatively low number of HSPCs in CB. Findings increasingly suggest that MSC-derived microvesicles (MSC-MVs) play an important role in the biological functions of their parent cells. We speculate that MSC-MVs may recapitulate the hematopoiesis-supporting effects of their parent cells. In the current study, we found MSC-MVs containing microRNAs that are involved in the regulation of hematopoiesis. We also demonstrated that MSC-MVs could improve the expansion of CB-derived mononuclear cells and CD34+cells and generate a greater number of primitive progenitor cells in vitro. Additionally, when MSC-MVs were added to the CB-MSC coculture system, they could improve the hematopoiesis-supporting effects of MSCs. These findings highlight the role of MSC-MVs in the ex vivo expansion of CB, which may offer a promising therapeutic approach in CB transplantation.

Activated Peripheral Mononuclear Cells Plus Soluble Plasma Tissue Factor Activate Factors VII and X in Cardiac Surgical Wounds.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 806-806
Author(s):  
Robert W. Colman ◽  
Mohammad M.H. Khan ◽  
Takashi Hattori ◽  
L. Henry Edmunds4
Keyword(s):  
Cardiac Surgery ◽  
Tissue Factor ◽  
Thrombin Generation ◽  
Mononuclear Cells ◽  
Factor Vii ◽  
Activate Factor ◽  
Factor X ◽  
Complex Wound ◽  
Activated Monocytes

Abstract Objectives: This study examines the role of pericardial wound monocytes in thrombin generation during clinical cardiac surgery with cardiopulmonary bypass (CPB). Background: The mechanism by which wound mononuclear cells rapidly express procoagulant activity is unexplained. Methods: Factor VII activation (FVIIa) was measured using recombinant, truncated, soluble tissue factor (rsTF) and various blood cells in vitro. FVIIa was also measured with monocytes and soluble plasma tissue factor taken before CPB and simultaneously from the pericardial wound and perfusion circuit during CPB in thirteen patients. Results: RsTF in combination with monocytes, but not platelets, neutrophils or red cells, accelerates activation of FVII beginning at 1 pmole/L rsTF. Less than 1% rsTF is bound, yet catalytic activity peaks at 7 minutes and decays afterwards. In wound plasma, monocytes are activated (MCP-1 = 29.5 ± 2.1 pmoles/L) and wound plasma tissue factor (wpTF) is substantially elevated (3.64 ± 0.45 pmoles/L) with 81.7% in the supernatant and 18.3% in microparticles. By Western blot all forms of plasma TF migrate at Mr 65 kDa [TF/FVII(FVIIa) complex]. Wound monocytes and C5a activated prebypass or perfusate monocytes plus wpTF convert all available FVII to FVIIa. Activated monocytes plus supernatant TF/FVII(VIIa) more efficiently activate factor X than microparticle TF/FVII(FVIIa). The correlation coefficient (r) between wound thrombin generation (F1.2) and wpTF is 0.944 (p = 0.0004). Conclusions: During clinical cardiac surgery with CPB wound monocytes plus wpTF or microparticle-free, protein fragments of wound tissue factor preferentially accelerate activation of FVII and FX. This system represents a new mechanism of thrombin generation.

Selective Increases of Procoagulant Activity in Rabbit Lymphoid Populations In Vitro Following Stimulation with Endotoxin: Dependence on Anatomic Source

1984 ◽  
Vol 51 (02) ◽  
pp. 228-231 ◽  
Author(s):  
Maria P McGee ◽  
Henry Rothberger ◽  
Tung-Kuang Lee
Keyword(s):  
Lymph Node ◽  
Tissue Factor ◽  
Mononuclear Cells ◽  
Procoagulant Activity ◽  
Mononuclear Leukocytes ◽  
Cell Populations ◽  
Factor Activity ◽  
Tissue Factor Activity

SummaryRabbit mononuclear leukocytes isolated from a variety of anatomic sites were examined for ability to generate procoagulant activity in vitro. Marrow, blood and spleen mononuclear cell populations were found to differ functionally from lymph node, thymus and alveolar populations by having much greater ability to increase in tissue factor activity in response to an endotoxin stimulus. Thus, after incubation in the presence of endotoxin, leukocytes obtained from marrow, blood, and spleen were found to increase in procoagulant activity characterized as tissue factor by 832, 1942 and 12.6 fold, respectively. In contrast, pulmonary alveolar macrophages increased in tissue factor activity only by 2.8 fold, and lymph node and thymus mononuclear cells showed little or no increases. These functional differences, demonstrated by exposing the six cell populations to endotoxin under controlled conditions in vitro, likely explain the similar pattern of anatomic selectivity of leukocyte tissue factor increases reported to occur in vivo during endotoxemia and Shwartzman reactions (1).

TLR Agonist Induced Cytokine Production in Human Multipotent Mesenchymal Stromal Cells: A Potential Mechanism How Hematopoiesis Is Enhanced during Generalized Inflammatory Conditions.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1411-1411
Author(s):  
Steffen Boettcher ◽  
Patrick Ziegler ◽  
Markus G. Manz
Keyword(s):  
Bone Marrow ◽  
Immune Responses ◽  
Cord Blood ◽  
Stromal Cells ◽  
Multipotent Mesenchymal Stromal Cells ◽  
Potential Mechanism ◽  
Gm Csf ◽  
Inflammatory Conditions ◽  
Cd34 Cells

Abstract Toll-like receptors (TLRs) function as receptors for different conserved pathogen associated products as well as certain host derived molecules. TLRs are expressed in several hematopoietic and non-hematopoietic cells. Their activation plays a key role in innate and adaptive immune responses to infectious agents, as well as in the development of pathologic conditions like tissue damage and cancer. Human multipotent mesenchymal stromal cells (MSCs) have been shown to differentiate into various mesenchymal tissues such as bone, cartilage, and fat, as well as marrow and lymphoid organ stroma cells. Human MSCs are able to maintain CD34+ cells to some extent in vitro. Furthermore, it has been demonstrated that upon intra bone marrow transplantation into adult immunodeficient mice MSC derived cells support human hematopoiesis in vivo. We hypothesized that MSCs express TLRs and are capable to respond to TLR agonists by changing their cytokine expression pattern in order to more efficiently support hematopoiesis according to respective needs in inflammatory conditions. MSCs from human bone marrow, cord blood, and umbilical cord whartons jelly were cultured by plastic adherence in IMDM 20% FCS, 1–8M dexamethasone (only during first 3 weeks), expanded for 2 passages, and subsequently analyzed. MSCs expressed gene-transcripts for IL-6, IL-7, IL-11, IL-15, SCF, TPO, FLT3L, M-CSF, GM-CSF, LIF, and SDF-1, while G-CSF was rarely detectable. Consistently, respective cytokines were measured in supernatants at the following, declining levels (pg/ml): IL-6 (10000–10E6) > SDF-1 > IL-11 > M-CSF > IL-7 > LIF > SCF, whereas GM-CSF was rarely detectable, G-CSF, FLT3-L, and TPO were not detectable by ELISA. MSCs were further analyzed for expression of TLRs by semiquantitative RT-PCR. TLR 1, 3, 4, 5, 6, and 9 expression, but not TLR 2, 7, 8, and 10 expression was detectable. Compared to human conventional (BDCA-1+, CD14−, CD19−) and plasmacytoid (BDCA-4+,CD14−,CD19−) blood dendritic cells, MSCs expressed TLR-3 and TLR-4 at levels up to 2 log higher than did conventional DCs, while TLR 9 expression was low. Upon in vitro stimulation with LPS (TLR-4 agonist), MSCs produced previously undetectable G-CSF and GM-CSF, and M-CSF levels increased about 4 fold compared to untreated MSCs, whereas stimulation with CpG motifs (TLR-9 agonists) did not lead to changes in cytokine release detected in supernatants. In co-culture experiments using MSCs as a feeder layer for cord blood CD34+ cells, MSCs supported hematopoiesis and the development of myeloid cells. Importantly, MSC preconditioning with LPS led to an 1,7 fold increase in total hematopoietic cell number, while preconditioning with CpG had no measurable effect. These results demonstrate that MSCs express certain TLRs, and are capable to respond to pathogen associated molecules with an increase in secretion of hematopoiesis-relevant cytokines, and thus reveal a potential mechanism how hematopoiesis is enhanced during generalized inflammatory conditions, supporting e.g. in case of gram-negative sepsis efficient innate immune responses.

A New Procoagulant Mechanism Mediated by Fucoidans

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4420-4420 ◽  
Author(s):  
Michael Dockal ◽  
Erwin Panholzer ◽  
Rudolf Hartmann ◽  
Hartmut J. Ehrlich ◽  
Friedrich Scheiflinger
Keyword(s):  
Tissue Factor ◽  
Thrombin Generation ◽  
Coagulation Factor ◽  
Coagulation Factors ◽  
Procoagulant Activity ◽  
Sulfated Polysaccharides ◽  
Intrinsic Pathway ◽  
In Vitro Characterization ◽  
Inhibitory Antibodies

Abstract Abstract 4420 Fucoidans are heterogeneous, polyanionic molecules with procoagulant activities in a wide concentration range. They have been described as non-anticoagulant sulfated polysaccharides (NASP) and shown to improve clotting in FVIII- and FIX-deficient plasma. In vitro characterization has suggested that fucoidans exert their procoagulant activity by inhibiting tissue factor pathway inhibitor (Liu et al. Thromb Haemost 2006; 95:68) and by accelerating thrombin-dependent FVa formation (Mutch et al. J Thromb Haemost 2007; 5 Suppl2). In our study we describe a new, previously unrecognized mechanism by which fucoidans act as procoagulant agents. The procoagulant activity of several fucoidans was characterized by calibrated automated thrombography in tissue factor (TF)-dependent experiments and by using coagulation factor-deficient plasmas. Spiking experiments with purified coagulation factors or inhibitory antibodies verified the mechanism. Stimulation of thrombin generation (TG) by fucoidans requires anionic lipid surfaces like synthetic phospholipid vesicles which contain phosphatidylserine and is TF-dependent (0-20pM). However, stimulatory activity was most pronounced in the absence of TF. Control experiments with corn trypsin inhibitor or FXII-deficient plasma excluded any involvement of the contact system. Plasmas from patients with congenital coagulation factor deficiencies were screened for TG to identify the target coagulation factor by which fucoidans exert their procoagulant activities. In the absence of TF, plasmas deficient in coagulation factors from the common pathway do not support fucoidan-mediated thrombin generation, whereas FVII-deficient plasma does. FXI was identified as the most upstream factor of the intrinsic pathway which is required for fucoidan-stimulated thrombin generation, suggesting it to be the target for the procoagulant activities of fucoidan. Spiking 30nM FXI to FXI-deficient plasma restored fucoidan-mediated TG and addition of polyclonal FXI inhibitory antibodies to normal plasma abrogated TG. Fucoidan-dependent TG did not improve when FXIa (60pM) was added to FXI-deficient plasma, suggesting activation of FXI by fucoidan. The relevance of this mechanism in hemophilia A plasma was studied by addition of low levels of FVIII (0.2-10%) resulting in a FVIII concentration-dependent increase in fucoidan-mediated TG. These results highlight the requirement of a functional intrinsic pathway for this new mechanism of fucoidans. Our findings present FXI activation at low TF concentrations as a possible mechanism for fucoidan. Disclosures: Dockal: Baxter Innovations GmbH: Employment. Panholzer:Baxter Innovations GmbH: Employment. Hartmann:Baxter Innovations GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment.

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