CLL-Derived Exosomes Educate Endothelial Cells to Become CLL-Supportive Cells

Background: CLL is characterized by a gradual accumulation of mature appearing long-lived lymphocytes. Several pro-survival pathways that protect CLL cells from apoptosis are activated in these cells. For example, IL-6 dependent phosphorylation of the signal transducer and activator of transcription 3 (STAT3) provides CLL cells with a survival and a proliferative advantages. What is the source of IL-6 is currently unknown. Within lymphoid organs CLL cells engage in complex molecular interactions. In these sites, CLL cells are not simply the seeds that grow on the supportive soil of the microenvironment but play an active role in shaping its surrounding. Secreted by all types of cells, exosomes are nano-scaled particles that travel in blood and function as stable intercellular transport vehicles that deliver their cargo to cells that engulf them. For example, CLL-derived exosomes (herein CLL-exosomes) are taken up by mesenchymal stromal cells, transforming them to cancer associated fibroblasts. Given the appropriate stimulation, endothelial cells produce IL-6. Therefore, we hypothesized that CLL-exosomes actively recruit endothelial cells to become IL-6-secreting cells.Methods: CLL cells from 37 patients were included in this study. CLL-exosomes were isolated by ultracentrifugation. Electron microscopy, NanoSight tracking analysis, flow cytometry and Western immunoblotting were used to characterize CLL exosomes. Exosomal uptake by HUVECs was assessed by flow cytometry and fluorescent microscopy. The phosphor-protein profiling of exposed HUVECs was analyzed by mass spectrometry. RT-PCR and Western immunoblotting were used to determine the expression profile of HUVEC-exposed cells. HUVECs were transfected with β-catenin containing plasmid using DNA transfection reagent. Cytokine levels were determined by ELISA and CHIP assay was used to identify activated transcription factors .Results: First, we isolated CLL-exosomes from 37 treatment naïve patients. For that purpose we grew CLL cells and collected the secreted exosomes after 72 hours by ultracentrifugation. By NanoSight tracking system we identified large amount of exosomes and verified the presence of the typical cap shaped vesicles by electron microscopy. Western immunoblotting confirmed the presence of CD63 and CD81 exosomal markers and by flow we detected CD19/CD5 on these particles. Next, we exposed HUVECs to an increasing amount of CLL-exosomes and verified by two distinct methods that these particles are up taken by HUVECs in a dose- and time- dependent manner. By mass spectrometry we found 53 phosphorproteins that were at least 2 folds upregulated and none that were downregulated in HUVEC-exposed cells. Pathway analysis unraveled the central position of β-catenin. Immunoprecipitation studies verified that levels of phosphor-β-catenin are upregulated while levels of total β-catenin remained unchanged, suggesting that CLL-exosomes induced phosphorylation rather than the generation of newly formed β-catenin and leaving us wondering whether upregulation of phosphor β-catenin is somehow beneficial to their parental neoplastic cells. Because the IL-6 promoter binds 3 transcription factors that are activated by β-catenin we assumed that by activating the β-catenin pathway, endothelial cells become "micro factories" for the production of humoral IL-6. To verify the role of β-catenin in promoting production of IL-6 molecules in endothelial cells we transfected HUVECs with β-catenin containing plasmid. By Western immunoblotting we verified that the protein levels of β-catenin were upregulated. Then, by ChIP assay we found that 3 different transcription factors, namely LEF/TCF, CEBP and NFkB increased their binding to the IL-6 promoter region by 1.5 to 12 folds in HUVECs that were transfected with β-catenin ORF. Finally we show that intracellular STAT3 of CLL cells that were grown on this IL-6-rich medium are phosphorylated on tyrosine residues and that the rate of CLL cells in active apoptosis was markedly decreased. Conclusions: CLL cells shape their own fate. They do so by sending exosomes that activate the β-catenin axes. In this way CLL cells reprogram endothelial cells to become IL-6 producing cells and IL-6 contributes to CLL cells' survival. Disclosures No relevant conflicts of interest to declare.

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Pleiotropic effects of laminar flow and statins depend on the Krüppel-like factor-induced lncRNA MANTIS

European Heart Journal ◽

10.1093/eurheartj/ehz393 ◽

2019 ◽

Vol 40 (30) ◽

pp. 2523-2533 ◽

Cited By ~ 22

Author(s):

Matthias S Leisegang ◽

Sofia-Iris Bibli ◽

Stefan Günther ◽

Beatrice Pflüger-Müller ◽

James A Oo ◽

...

Keyword(s):

Endothelial Cells ◽

Transcription Factors ◽

Laminar Flow ◽

Therapeutic Potential ◽

Statin Treatment ◽

Telomerase Activity ◽

Dependent Manner ◽

Protective Effects ◽

Monocyte Adhesion ◽

Beneficial Effects

Abstract Aims To assess the functional relevance and therapeutic potential of the pro-angiogenic long non-coding RNA MANTIS in vascular disease development. Methods and results RNA sequencing, CRISPR activation, overexpression, and RNAi demonstrated that MANTIS, especially its Alu-element, limits endothelial ICAM-1 expression in different types of endothelial cells. Loss of MANTIS increased endothelial monocyte adhesion in an ICAM-1-dependent manner. MANTIS reduced the binding of the SWI/SNF chromatin remodelling factor BRG1 at the ICAM-1 promoter. The expression of MANTIS was induced by laminar flow and HMG-CoA-reductase inhibitors (statins) through mechanisms involving epigenetic rearrangements and the transcription factors KLF2 and KLF4. Mutation of the KLF binding motifs in the MANTIS promoter blocked the flow-induced MANTIS expression. Importantly, the expression of MANTIS in human carotid artery endarterectomy material was lower compared with healthy vessels and this effect was prevented by statin therapy. Interestingly, the protective effects of statins were mediated in part through MANTIS, which was required to facilitate the atorvastatin-induced changes in endothelial gene expression. Moreover, the beneficial endothelial effects of statins in culture models (spheroid outgrowth, proliferation, telomerase activity, and vascular organ culture) were lost upon knockdown of MANTIS. Conclusion MANTIS is tightly regulated by the transcription factors KLF2 and KLF4 and limits the ICAM-1 mediated monocyte adhesion to endothelial cells and thus potentially atherosclerosis development in humans. The beneficial effects of statin treatment and laminar flow are dependent on MANTIS.

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Proeryptotic Activity of 4-Hydroxynonenal: A New Potential Physiopathological Role for Lipid Peroxidation Products

Biomolecules ◽

10.3390/biom10050770 ◽

2020 ◽

Vol 10 (5) ◽

pp. 770

Author(s):

Mario Allegra ◽

Ignazio Restivo ◽

Alberto Fucarino ◽

Alessandro Pitruzzella ◽

Sonya Vasto ◽

...

Keyword(s):

Electron Microscopy ◽

Lipid Peroxidation ◽

Scanning Electron Microscopy ◽

Flow Cytometry ◽

Endothelial Cells ◽

Caspase 3 ◽

Morphological Alterations ◽

Signalling Molecule ◽

Free Haemoglobin ◽

Scanning Electron

Background: Eryptosis is a physiological, apoptosis-like death of injured erythrocytes crucial to prevent premature haemolysis and the pathological sequalae generated by cell-free haemoglobin. When dysregulated, the process is associated to several inflammatory-based pathologies. 4-Hydroxy-trans-2-nonenal (HNE) is an endogenous signalling molecule at physiological levels and, at higher concentrations, is involved in the pathogenesis of several inflammatory-based diseases. This work evaluated whether HNE could induce eryptosis in human erythrocytes. Methods: Measurements of phosphatidylserine, cell volume, intracellular oxidants, Ca++, glutathione, ICAM-1, and ceramide were assessed by flow cytometry. Scanning electron microscopy evaluated morphological alterations of erythrocytes. Western blotting assessed caspases. PGE2 was measured by ELISA. Adhesion of erythrocytes on endothelial cells was evaluated by gravity adherence assay. Results: HNE in the concentration range between 10–100 µM induces eryptosis, morphological alterations correlated to caspase-3 activation, and increased Ca++ levels. The process is not mediated by redox-dependent mechanisms; rather, it strongly depends on PGE2 and ceramide. Interestingly, HNE induces significant increase of erythrocytes adhesion to endothelial cells (ECs) that are in turn dysfunctionated as evident by overexpression of ICAM-1. Conclusions: Our results unveil a new physiopathological role for HNE, provide mechanistic details of the HNE-induced eryptosis, and suggest a novel mechanism through which HNE could exert pro-inflammatory effects.

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Clarithromicin Induces Autophagy in Myeloma Cells.

Blood ◽

10.1182/blood.v108.11.3509.3509 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3509-3509 ◽

Cited By ~ 2

Author(s):

Miki Nakamura ◽

Takahiro Kamimoto ◽

Tamotsu Yoshimori ◽

Hiroaki Mitsuya ◽

Hiroyuki Hata

Keyword(s):

Electron Microscopy ◽

Growth Inhibition ◽

Cell Lines ◽

Fluorescent Microscopy ◽

Myeloma Cell ◽

Pi3 Kinase ◽

Dependent Manner ◽

Myeloma Cells ◽

Akt Phosphorylation ◽

Dose Dependent

Abstract Introduction Some macrolide antibiotics exert effects other than anti-bacterial activity on the growth and viability of certain cancer cells. The presence of cytoplasmic vacuoles is one the salient features of autophagy, a cellular event believed to recycle cellular ingredients under nutrient-starved conditions. Such vacuoles (autophagosomes) fuse with lysozomes, generating autolysozomes toward later stages of autophagy, digesting organelles and degenerated proteins. Our own and others’ findings that a macrolide antibiotic clarithromicin (CAM) occasionally shows anti-myeloma effects when combined with thalidomide and/or dexamethasone prompted us to examine CAM for its effects on myeloma cells in vitro. Methods Four myeloma cell lines (12PE, KHM-11, KMM-1 and U266) and primary myeloma cells purified by CD138-conjugated immune-magnetic beads (Miltenvi Biotec, Auburn, CA) were utilized. Clarithromicin was obtained from Taisho-Toyama pharmaceuticals (Tokyo, JAPAN). Morphology was analyzed either by May-Giemza staining or electron microscopy. Autolysozome was stained with Lysotracker (Invitrogen, Carlsbad, CA) and analyzed using fluorescent microscopy. Antibody to LC3 was obtained from Dr. T. Yoshimori (Department of Cellular Regulation, Research Institute for Microbial Diseases, Osaka University). Results and discussion CAM induced vacuoles in the cytoplasm of both myeloma cell lines and primary myeloma cells at concentrations ranging from 10 to 50 mg/ml at a dose-dependent manner after ~18 hours treatment. Electron microscopy revealed that those vacuoles morphologically resemble autolysozomes. To further confirm the identity of autolysozomes, cells were stained with Lysotracker, which specifically stains acid lysozome. After the treatment with CAM, the accumulation of vacuoles in the cytoplasm, stained with Lysotacker, was observed. Since initiation of autophagy depends on PI3-kinase, we investigated whether CAM induced AKT phosphorylation. AKT phosphorylation was readily observed, and moreover, the emergence of vacuoles stainable with Lysotracker was inhibited when the cells were pretreated with PI3-kinase inhibitors, 3MA or LY294002, strongly suggesting that vacuolation is indeed mediated with PI3-kinase. To further confirm that autopahgy is induced by CAM, the process of LC3-I to LC3-II, a hallmark of autophagy, was examined. We found that the induction of LC3-II by CAM occurred at a dose-dependent manner. Taken together, these findings strongly suggest that CAM induces autolysozome accumulation through activating PI3-kinase. Finally, we examined whether CAM induced apoptosis when combined with thalidomide. Three myeloma cells lines, which abundantly expressed Bcl-2, showed no growth inhibition, while KHM-11, which was defective in Bcl-2, showed marked apoptosis and growth inhibition with the combination of CAM and thalidomide, suggesting that CAM might potentially augment anti-myeloma activity of thalidomide although the mechanisms are to be determined. Taken these observations together, the manipulation of certain autophagy processes with reagents such as macrolides (i.e., CAM) might represent a new therapeutic approach in the treatment of myeloma. We hypothesize that CAM dually functions in the event of autophagy, i.e., it initiates autophagy while it suppresses autophagy at later stages. Further study under the hypothesis is currently underway.

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Heme Induces Endothelial Tissue Factor Expression: Potential Role in Hemostatic Activation in Patients with Intravascular Hemolysis Including Sickle Cell Disease.

Blood ◽

10.1182/blood.v112.11.1902.1902 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1902-1902

Author(s):

Yamaja Setty ◽

Suhita Gayen Betal ◽

Jie Zhang ◽

Nigel S Key ◽

Marie Stuart

Keyword(s):

Flow Cytometry ◽

Endothelial Cells ◽

Sickle Cell Disease ◽

Sickle Cell ◽

Whole Blood ◽

Dependent Manner ◽

Cell Disease ◽

Intravascular Hemolysis ◽

Tnf Α ◽

Endothelial Tissue

Abstract Plasma levels of heme in the 20 to 600 μM range are found in clinical conditions associated with intravascular hemolysis including paroxysmal nocturnal hemoglobinuria and sickle cell disease, conditions also associated with a thrombotic tendency. Objectives: To investigate whether heme, an inflammatory mediator and a product of intravascular hemolysis in patients with hemolytic anemia including sickle cell disease (SCD), could modulate hemostasis by an effect on endothelial tissue factor (TF) expression. Additionally, in SCD patient-related studies, we assessed whether any association existed between whole blood TF activity (WBTF) and levels of surrogate markers of intra-vascular hemolysis including lactate dehydrogenase (LDH) and reticulocyte counts. Methods: Following incubation of human endothelial cells (from umbilical vein and/or lung microvasculature) with heme (1 to 100 μM) for various times (30 minutes to 8 hours), levels of TF protein were assessed using ELISA, flow cytometry and/or Western blotting; and TF mRNA by a semi-quantitative RT-PCR. An assay for TF functional activity was performed using a chromogenic tenase activity kit where specificity of TF activity was tested in antibody-blocking experiments. Three TF-specific antibodies including a rabbit polyclonal and two mouse monoclonal (clones hTF-1 and TF9-10H10) antibodies were used in assays involving TF protein analysis. All experiments were performed in media containing polymyxin B to neutralize any potential endotoxin contamination. In patient-related studies, 81 subjects with SCD (1 to 21 years) were evaluated for levels of WBTF, LDH, and reticulocyte counts and data analyzed for potential relationships. Results: Heme induced TF protein expression on the surface of both macro- and micro-vascular endothelial cells in a concentration-dependent manner with 12- to 50-fold induction noted (ELISA assays) between 1 and 100 μM heme (P<0.05, n=3 to 6). Complementary flow cytometry studies showed that the heme-mediated endothelial TF expression was quantitatively similar to that induced by the cytokine TNF-α. Heme also up-regulated endothelial expression of TF mRNA (8- to 26-fold, peak expression at 2 hours postagonist treatment), protein (20- to 39-fold, peak expression at 4 hours) and procoagulant activity (5- to 13-fold, peak activity at 4 hours post-agonist treatment) in a time-dependent manner. Time-course of heme-mediated TF antigen expression paralleled induction of procoagulant activity with antibody blocking studies demonstrating specificity for TF protein. Potential involvement of endogenously released cytokines including IL-1α and TNF-α in mediating the heme effect was next explored. We found that the latter cytokines are not involved, since antibodies against IL-1α and TNF-α, and an IL-1- receptor antagonist failed to block heme-induced endothelial TF expression. Inhibition of heme-induced TF mRNA expression by sulfasalazine and curcumin suggested that the transcription factor NFκB was involved in mediating heme-induced effect. In patient-related studies, whole blood TF levels in SCD correlated positively with both LDH (r=0.72, p<0.000001), and reticulocyte count (r=0.60, p<0.000001). Conclusions: Our findings demonstrate that heme induces TF expression in endothelial cells, and that the observed effects occurred at patho-physiologically relevant heme concentrations. Our results suggest that heme-induced endothelial TF expression may provide a pathophysiologic link between the intravascular hemolytic milieu and the hemostatic perturbations previously noted in patients with hemolytic anemia including sickle cell disease.

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Oxyhemoglobin-induced apoptosis in cultured endothelial cells

Journal of Neurosurgery ◽

10.3171/jns.1999.91.3.0459 ◽

1999 ◽

Vol 91 (3) ◽

pp. 459-465 ◽

Cited By ~ 49

Author(s):

Kotaro Ogihara ◽

Alexander Y. Zubkov ◽

David H. Bernanke ◽

Adam I. Lewis ◽

Andrew D. Parent ◽

...

Keyword(s):

Electron Microscopy ◽

Transmission Electron Microscopy ◽

Endothelial Cells ◽

Time Dependent ◽

Dependent Manner ◽

Content Type ◽

Aortic Endothelial Cells ◽

Bovine Aortic Endothelial Cells ◽

Transmission Electron ◽

Fine Print

Object. Oxyhemoglobin (OxyHb) is one of the most important spasmogens for cerebral vasospasm that follows aneurysmal subarachnoid hemorrhage. The cytotoxic effect of OxyHb has been documented in endothelial and smooth-muscle cells; however, the pattern of cell death—necrosis or apoptosis—as the final stage of cell damage has not been demonstrated. This study was undertaken to determine if OxyHb induces apoptotic changes in cultured bovine aortic endothelial cells.Methods. Confluent bovine aortic endothelial cells were treated with OxyHb in a concentration- and time-dependent manner. Cell density was assayed by counting the number of cells that attached to culture dishes after exposure to OxyHb. To identify apoptotic changes, the investigators used three specific methods: DNA fragmentation (electrophoreses), the apoptotic body (transmission electron microscopy), and cleavage of poly (adenosine diphosphate ribose) polymerase (PARP [Western blotting]).Conclusions. Oxyhemoglobin decreased cell density in a concentration- and time-dependent manner. Analysis of DNA showed a pattern of internucleosomal cleavage characteristic of apoptosis (DNA ladder). Transmission electron microscopy demonstrated condensation of nuclei and apoptotic bodies in OxyHb-treated endothelial cells. Western blotting with the PARP antibody revealed that the 116-kD PARP was cleaved to the 85-kD apoptosis-related fragment. These results for the first time demonstrated that the OxyHb induces apoptosis in cultured endothelial cells.

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Brain-Derived Microparticles Induce Systemic Coagulation Associated with Traumatic Brain Injury

Blood ◽

10.1182/blood.v124.21.1497.1497 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1497-1497

Author(s):

Jing-fei Dong ◽

Ye Tian ◽

Breia Salsbery ◽

Hengjie Yuan ◽

Min Wang ◽

...

Keyword(s):

Electron Microscopy ◽

Traumatic Brain Injury ◽

Flow Cytometry ◽

Coagulation Factors ◽

Endothelial Barrier ◽

Dependent Manner ◽

Activated Platelets ◽

Dose Dependent ◽

Made In

Abstract Uncontrolled hemorrhage is a leading cause of the preventable deaths that occur in patients with trauma. The cause of trauma-associated coagulopathy is multifactorial, including blood loss, consumption of coagulation factors and platelets, the dilution of coagulation factors and platelets due to fluid resuscitation, and hypothermia. Traumatic brain injury (TBI) lacks two key causal factors for coagulopathy: heavy blood loss and a large volume of fluid resuscitation, but is associated with a significantly higher incidence of coagulopathy. The pathogenesis of this TBI-associated coagulopathy remains poorly understood. We tested the hypothesis that brain-derived microparticles (BDMPs) released from an injured brain play a causal role in developing systemic coagulopathy after TBI. Here, we report that mice subjected to fluid percussion injury (1.9±0.1 atm) developed a BDMP-dependent hypercoagulable state, with a peak level of plasma glial cell and neuronal microparticles, reaching 17,496 ± 4,833/µl and 18,388 ± 3,657/µl 3 hrs after TBI. BDMPs were measured by flow cytometry using triple gating based on particle size and the expression of neural cell markers and phosphatidylserine (PS). To exclude contributions to the coagulopathy of non-neural cell microparticles released during trauma stress, BDMPs were made from normal brain by freeze-thawing and mechanical injury. BDMPs thus made had below detection levels of microparticles from leukocytes (CD45), endothelial cells (CD144), erythrocytes (CD235a), and platelets (CD42b). Uninjured mice injected with BDMPs made in vitro developed a hyper-turn-hypo-coagulable state in a dose-dependent manner as measured by the rates of clot formation and fibrinogen depletion, resulting in microvascular fibrin deposition in the lungs, kidney and heart. BDMPs measured 50 – 500 nm with relatively intact membranes under transmission electron microscopy and expressed neuronal or glial cell markers and procoagulant PS and tissue factor (TF). BDMPs promoted clot formation in a PS-dependent assay at a maximal activity of ~1 x 105 BDMPs/µl, equivalent to 1.6 µg/µl of purified brain PS. They were equally active in promoting thrombin generation in a PS-and TF-dependent manner, BDMPs at 2.5 x 104 /µl yielding an activity equivalent to 1 pM of soluble TF. The procoagulant activity of BDMPs was significantly stronger than microparticles generated from collagen-stimulated platelets and was blocked by the PS-binding lactadherin in a dose-dependent manner. Consistent with observations made in the mouse models, fetal hippocampal cells in culture produced microparticles upon injury. These microparticles transmigrated through the disrupted endothelial barrier in the presence of live, but not lyophilized platelets. BDMP-bound platelets were detected by flow cytometry and scan electron microscopy. They activated platelets as measured by increases in calcium influx and CD62p expression, but did not induce platelet aggregation directly or in the presence of low doses of collagen. In summary, we have studied acute changes in coagulation associated with TBI using a mouse FPI model combined with in vitro experiments. Focusing on the first 6 hrs post-TBI minimizes confounding changes induced by secondary events, such as ischemic injury. The results define a causal role for BDMPs in the TBI-associated systemic coagulation. We also show that BDMPs activated platelets. Activated platelets may facilitate the transmigration of BDMPs through the disrupted endothelial barrier by releasing pro-inflammatory mediators to promote local inflammation at a site of vascular injury. This notion is supported by the finding that live, but not lyophilized platelets and, to lesser degree, plasma from activated platelets promoted BDMP transmigration through a monolayer of endothelial cells. Finally, the PS binding lactadherin blocked the BDMP-dependent procoagulant activity, raising two interesting perspectives. First, PS scavengers and neutralizing molecules may reduce or prevent coagulopathy associated with TBI. Second, an intrinsic or acquired deficiency in the PS-dependent clearance of microparticles may predispose an individual to consumptive coagulopathy associated with TBI and other conditions. Disclosures No relevant conflicts of interest to declare.

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Abstract 267: Opposing Functions of Two Splice Variants of the Human Transcription Factor Grainyhead-like 3 in Endothelial Cells And in vivo

Circulation Research ◽

10.1161/res.113.suppl_1.a267 ◽

2013 ◽

Vol 113 (suppl_1) ◽

Author(s):

Joachim Altschmied ◽

Nicole Büchner ◽

Sascha Jakob ◽

Sabrina Farrokh ◽

Christine Goy ◽

...

Keyword(s):

Endothelial Cells ◽

Transcription Factor ◽

Transcription Factors ◽

Target Genes ◽

Splice Variants ◽

Luciferase Reporter ◽

Dependent Manner ◽

Active Transcription ◽

Human Transcription Factor

Grainyhead-like 3 (GRHL3) is a member of the evolutionary conserved Grainyhead family of transcription factors. In humans, three isoforms are derived from differential first exon usage and alternative splicing, which differ only in their N-terminus. Isoform 2, the only variant also present in mouse, is required for endothelial cell (EC) migration and protects against apoptosis. The functions of the human specific isoforms 1 and 3, which are derived from an alternatively spliced pre-mRNA, have not yet been investigated, although all three isoforms are expressed in EC. Therefore, we have assessed their effects on EC migration and apoptosis. Overexpression of the two proteins had opposite effects on EC migration, with isoform 1 acting pro-migratory. This protein also protected EC against apoptosis in an eNOS-dependent manner, whereas isoform 3 had no effect. These opposing outcomes with respect to apoptosis EC were corroborated by isoform-specific knockdowns. With reporter assays using a GRHL3-specific luciferase reporter we demonstrated that both are active transcription factors. Microarray analyses revealed that they induce divergent target gene sets in EC. Two validated targets, Akt2 and Mxi1, which are upregulated by isoform1, are regulators of Akt1-, and thus eNOS-phosphorylation and apoptosis, which could explain the effects of this protein on these processes. In vivo, overexpression of isoform 3 in zebrafish embryos resulted in increased lethality and severe deformations, while isoform 1 had no deleterious effect. In conclusion, our data demonstrate that the splice variant derived isoforms 1 and 3 of the human transcription factor GRHL3 induce opposing effects in primary human endothelial cells and in a whole animal model, most likely through the induction of different target genes.

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Circulating CD62E Occurs in Soluble Form as Well as Bound to Endothelial Microparticles (EMP): Functional Differences in Platelet Aggregation in Thrombotic Thrombocytopenic Purpura (TTP).

Blood ◽

10.1182/blood.v104.11.2624.2624 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2624-2624

Author(s):

Joaquin J. Jimenez ◽

Wenche Jy ◽

Lucia M. Mauro ◽

Michael N. Markou ◽

George W. Burke ◽

...

Keyword(s):

Flow Cytometry ◽

Endothelial Cells ◽

Platelet Aggregation ◽

Acute Phase ◽

Critical Role ◽

In Vivo Studies ◽

Soluble Form ◽

Dependent Manner

Abstract Injured endothelial cells (EC) are believed to play a critical role in the pathophysiology of TTP. Soluble markers of endothelial disturbance measured by enzyme-linked immunoassay (ELISA) have been found elevated in TTP. We have recently demonstrated an increase in the release of CD31/42b- EMP, and CD62E+ EMP. Moreover, we have observed that CD62E+ EMP also express vWF. The aim of this study was to quantitate soluble (s) vs. EMP-bound CD62E (bCD62E) in vitro and in vivo, in relation to the functional activity of vWF+ EMP. METHODS: Brain and renal microvascular endothelial cells (MVEC) were cultured and treated with 10ng/mL TNF-α to induce activation, or deprived of serum and growth factors (GFD) to induce apoptosis. Culture supernatants were collected and evaluated in a time-dependent manner. For in vivo studies, platelet-poor plasma was obtained from 4 TTP patients during the acute phase and upon remission. Filtration through 0.1μm, which retains most EMP, was employed to discriminate between (s) and bCD62E. sCD62E was measured by ELISA post-filtration and bCD62E by ELISA pre-filtration. Additionally, CD62E+ and CD62E+/vWF+ EMP were measured by flow cytometry. To assess pro-aggregatory function, EMP were added to washed platelets in the presence of 1 mg/mL ristocetin and aggregates were measured by flow cytometry. RESULTS: In vitro: Activation did not induce release of sCD62E at 3 hours, although bCD62E was present (1.5±0.5X106 EMP/mL). At 6 hours, some sCD62E was detected in the filtrate (0.09±0.02 ng/mL), but most was present in the unfiltered medium (3.5±0.85 ng/mL), signifying that the majority was bCD62E, confirmed by a doubling of CD62E+ EMP (3.0±0.6X106/mL). Subsequently, sCD62E levels were 1.0±0.2 ng/mL at 12 hr, 3.5±0.7 ng/mL at 18 hr, and 5±0.9 ng/mL at 24 hr. In contrast, EMP counts at 12, 18 and 24 hours were 4.6±1, 7±1.3 and 9±1.8 X106/mL (p=0.01, p=0.01, p=0.02, respectively). For all time periods, 40-60% of CD62E were positive for vWF. In control or GFD cultures, there was not a significant increase in sCD62E or CD62E+ EMP at any time period. MVEC from renal gave similar results. In acute TTP plasma samples, CD62E measured by ELISA was significantly increased (65±22 ng/mL) vs. remission (30±6 ng/mL). bCD62E accounted for 50% in acute and 15% in remission. CD62E+/vWF+ EMP were significantly elevated in plasma from acute TTP patients vs. remission (15±4.5 vs. 3±0.5, p=0.01). Sample filtration resulted in a decrease of >95% EMP in both acute and remission TTP plasma. MVEC-derived CD62E+/vWF+ EMP resulted in a dose-dependent increase in platelet aggregation. Additionally, plasma from 4 TTP patients with elevated CD62E+/vWF+ EMP obtained during the acute phase enhanced the formation of platelet aggregates by 48±12% (p=0.02) above remission plasma with low EMP counts. CONCLUSIONS: The results demonstrate that CD62E heretofore regarded as a soluble marker of endothelial dysfunction, in reality exists in both a soluble and EMP-bound form. Indeed, this distinction is highly relevant because CD62E+ EMP also express vWF and are pro-aggregatory to platelets. These EMP have been shown to be elevated during the acute phase of TTP and decrease upon remission. Thus, CD62E+/vWF+ EMP may be active participants in the formation of platelet-rich thrombi in TTP.

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Endothelial Cell Secretion Combined with Shear Stress Strongly Increase Platelet Production by Human Megakaryocytes,

Blood ◽

10.1182/blood.v118.21.3411.3411 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3411-3411

Author(s):

Yasmine Ouzegdouh ◽

Laurence Momeux ◽

Claude Capron ◽

Elisabeth Cramer Bordé

Keyword(s):

Electron Microscopy ◽

Bone Marrow ◽

Flow Cytometry ◽

Endothelial Cells ◽

Shear Stress ◽

Conflicts Of Interest ◽

Video Microscopy ◽

Platelet Production ◽

Platelet Size ◽

Platelet Release

Abstract Abstract 3411 It is well established that megakaryocytes (MK), in order to achieve terminal maturation in the bone marrow, migrate from the osteoblastic niche to the vascular niche, close to medullary sinusoids. It is also admitted that, in order to release platelets, MK extend cytoplasmic extensions in the lumen of the sinusoid followed by platelet release. Therefore, we have investigated the role of endothelial cells combined with shear stress on MK late differentiation steps and platelet production. To do so, human MK were grown from umbilical cord blood or bone marrow CD34+ cells, in the presence of Stem Cell Factor (SCF) and thrombopoietin for 10 days. Then they were co-cultured with human endothelial cells (HUVEC) for 3 days and analysed by video-microscopy, flow cytometry, immunofluorescence and electron microscopy. In these conditions, most MK (>50%) extended numerous and prominent proplatelets: immunofluorescence showed virtually complete unwinding of their cytoplasm, which extended in the form of long proplatelets; this rarely occurred in control MK. Electron microscopy showed that these MK were formed by a coarse chromatin nucleus surrounded by a thin cytoplasmic ring and surrounded by platelet-size territories displaying alpha-granules, mitochondria and canalicular system which coincided to the sections of the proplatelet swellings and to the unwinding of the demarcation membrane system. Another type of experiment of co-culture MK/endothelial cells was conducted in transwells and led to similar results, ie, increase of MK cytoplasmic maturation and proplatelet formation indicating that cell/cell contact was not necessary for up-regulating proplatelet production, rather that (a) soluble product(s) was(ere) secreted from endothelial cells. Flow cytometry, at this step, failed to demonstrate significant changes in the platelet production rate. We then submitted MK co-cultured with endothelial cells to shear stress and examined them by flow cytometry and video microscopy. We could then demonstrate that platelet release was strongly increased (× 3.8 ± 0.9, n=3) when MK had been in contact with endothelial cells compared to control MK. The released platelet-size particles expressed CD41 and, when stimulated by thrombin, were also able to express CD62P. Video-microscopy confirmed that proplatelet and platelet shedding occurred after exposition of mature MK to shear stress. When examined by video-microscopy, live MK whose DNA had been stained with the fluorescent dye Hoechst 33342 showed that nuclear lobes separated under high shear stress: indeed they became located at opposite poles of the cell, while the cytoplasmic volume extended and elongated, becoming organized in proplatelets which exhibited a succession of platelet size subunits; eventually cytoplasmic scission occurred in parallel with MK nuclear lobe segregation in distinct cell fractions, each carrying proplatelets; proplatelets subsequently fractionated and were then released from the cell core containing the fluorescent nuclear lobe. Thus shear stress leads both to cytoplasm and nucleus fragmentation. This is a dynamic explanation to the fact that entire MK nuclei are rarely found in the human bone marrow. This observation also gives sense to the unique phenomenon of MK polyploidy. In conclusion, this study indicates that endothelial microenvironment combined with circulatory shear forces are determinant up-regulating factors which increase platelet production. It also shows that shear stress is able to induce nuclear as well as cytoplasmic MK fragmentation, leading to a new anatomical concept of circulating platelet shedding MK subunits. Disclosures: No relevant conflicts of interest to declare.

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p53 and c-Myc Are Critical Signaling Hubs That Maintain Chronic Myeloid Leukemia

Blood ◽

10.1182/blood.v122.21.1465.1465 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1465-1465

Author(s):

Sheela A Abraham ◽

Lisa Hopcroft ◽

Emma Carrick ◽

Andrew Williamson ◽

Andrew Pierce ◽

...

Keyword(s):

Mass Spectrometry ◽

Stem Cells ◽

Flow Cytometry ◽

Chronic Myeloid Leukemia ◽

Cell Expansion ◽

Myeloid Leukemia ◽

Dose Effect ◽

Dependent Manner ◽

Cd34 Cells ◽

Hub Proteins

Abstract Chronic myeloid leukemia (CML) is a clonal disorder of the hematopoietic system, leading to increased production of mature and progenitor myeloid cells. Although protein tyrosine kinase inhibitors (TKI) have been successful in managing the disease, there are exceptions where drug resistance and onset of blast crisis occur. Furthermore TKIs are ineffective against leukemic stem cells (LSC) that are responsible for disease initiation and maintenance. We have shown mRNA changes in primitive hematopoietic cells do not correlate directly to protein changes. Therefore to elucidate fundamental cellular differences between CML and normal cells we employed a proteomic approach (mass spectrometry with isobaric tagging for relative quantification). This approach permits unbiased analyses using direct comparative quantification of peptides and thus proteins from chronic phase CML and normal CD34+ human samples. Systematic data analysis identified that the majority of deregulated proteins are connected and regulated by two oncogenes with well defined roles in human disease, p53 and c-myc. The direction of regulation inferred suppression of p53 and up-regulation of c-myc. Altered expression of key proteins was validated using western blotting and immuno-fluorescence approaches. All (6/6) candidate/hub proteins identified using mass spectrometry were confirmed using these orthogonal approaches. Based on our systematic analysis, we targeted the candidate hubs using the drugs RITA (activates p53) and CPI-203 (inhibits c-myc expression; provided by Constellation Pharmaceuticals). In CML CD34+ cells, RITA reduced cell expansion in a concentration-dependent manner and induced significant levels of apoptosis as confirmed by positive staining of Annexin V and 4',6-diamidino-2-phenylindole (DAPI) using flow cytometry. CPI-203 also reduced cell expansion, but importantly induced differentiation in addition to apoptosis, as supported by flow cytometric monitoring of levels of carboxyfluorescein succinimidyl ester (CFSE) and CD34. Overlays of CFSE plots for untreated control vs. CPI-203 demonstrated that as cells divided in the presence of CPI-203, there was clear and rapid loss of CD34 expression which was not seen with RITA treatment. By measuring the dose-effect relationship of each drug alone and in combination, we demonstrated potent synergy with combination index (CI) values ranging from 0.034-0.286 based on loss of cell viability. Using flow cytometry we gated on CD34+38- CML cells to enable quantification of the differential effects of each drug alone and in combination against the most primitive and quiescent 1-5% of total CD34+ cells. Critically the apoptotic effect was inclusive of primitive CD34+38- cells and quiescent CFSEmax populations. In addition, experiments combining RITA and CPI-203 demonstrated undetectable colony forming cell units at the highest concentrations of drug used. Importantly, it appears that combining these two drugs has negligible effects on normal CD34+ cell counts, apoptosis and CFSE profiles. Currently NOD-SCID IL2R gamma null (NSG) repopulation assays are underway to determine if these drugs affect stem cells capable of engrafting immunocompromised mice. Our systems biology approach suggests that altered c-myc and p53 function underlie the most significant cellular differences within CML CD34+ cells, which has not been previously demonstrated. We confirm that in CML, p53 and c-myc hub proteins have the ability to modulate downstream defined target proteins thereby enhancing survival and proliferation and thus allowing maintenance of disease. Disclosures: No relevant conflicts of interest to declare.

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