Plasma microparticles of sickle patients during crisis or taking hydroxyurea modify endothelium inflammatory properties

Blood ◽  
2020 ◽  
Vol 136 (2) ◽  
pp. 247-256 ◽  
Author(s):  
Yohann Garnier ◽  
Séverine Ferdinand ◽  
Marie Garnier ◽  
Kizzy-Clara Cita ◽  
Régine Hierso ◽  
...  
Keyword(s):  
Steady State ◽  
Sickle Cell ◽  
Messenger Rna ◽  
Ex Vivo ◽  
Biological Effects ◽  
Annexin V ◽  
Neutrophil Adhesion ◽  
Dependent Manner ◽  
High Concentration ◽  
The Impact

Abstract Microparticles (MPs) are submicron extracellular vesicles exposing phosphatidylserine (PS), detected at high concentration in the circulation of sickle cell anemia (SS) patients. Several groups studied the biological effects of MPs generated ex vivo. Here, we analyzed for the first time the impact of circulating MPs on endothelial cells (ECs) from 60 sickle cell disease (SCD) patients. MPs were collected from SCD patients and compared with MPs isolated from healthy individuals (AA). Other plasma MPs were purified from SS patients before and 2 years after the onset of hydroxyurea (HU) treatment or during a vaso-occlusive crisis and at steady-state. Compared with AA MPs, SS MPs increased EC ICAM-1 messenger RNA and protein levels, as well as neutrophil adhesion. We showed that ICAM-1 overexpression was primarily caused by MPs derived from erythrocytes, rather than from platelets, and that it was abolished by MP PS capping using annexin V. MPs from SS patients treated with HU were less efficient to induce a proinflammatory phenotype in ECs compared with MPs collected before therapy. In contrast, MPs released during crisis increased ICAM-1 and neutrophil adhesion levels, in a PS-dependent manner, compared with MPs collected at steady-state. Furthermore, neutrophil adhesion was abolished by a blocking anti–ICAM-1 antibody. Our study provides evidence that MPs play a key role in SCD pathophysiology by triggering a proinflammatory phenotype of ECs. We also uncover a new mode of action for HU and identify potential therapeutics: annexin V and anti–ICAM-1 antibodies.

Increased Procoagulant Activity of Red Blood Cells from Patients with Homozygous Sickle Cell Disease and β-Thalassemia

1996 ◽  
Vol 76 (03) ◽  
pp. 322-327 ◽  
Author(s):  
Dominique Helley ◽  
Amiram Eldor ◽  
Robert Girot ◽  
Rolande Ducrocq ◽  
Marie-Claude Guillin ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Red Blood Cells ◽  
Sickle Cell ◽  
Blood Cells ◽  
Annexin V ◽  
Mean Value ◽  
Procoagulant Activity ◽  
Dependent Manner ◽  
Cell Disease ◽  
Homozygous Sickle Cell Disease

SummaryIt has recently been proved that, in vitro, red blood cells (RBCs) from patients with homozygous β-thalassemia behave as procoagulant cells. The procoagulant activity of β-thalassemia RBCs might be the result of an increased exposure of procoagulant phospholipids (i. e. phosphatidylserine) in the outer leaflet of the membrane. In order to test this hypothesis, we compared the catalytic properties of RBCs of patients with β-thalassemia and homozygous sickle cell disease (SS-RBCs) with that of controls. The catalytic parameters (Km, kcat) of prothrombin activation by factor Xa were determined both in the absence and in the presence of RBCs. The turn-over number (kcat) of the reaction was not modified by normal, SS- or (3-thalassemia RBCs. The Km was lower in the presence of normal RBCs (mean value: 9.1 µM) than in the absence of cells (26 µM). The Km measured in the presence of either SS-RBCs (mean value: 1.6 µM) or β-thalassemia RBCs (mean value: 1.5 pM) was significantly lower compared to normal RBCs (p <0.001). No significant difference was observed between SS-RBCs and p-thalassemia RBCs. Annexin V, a protein with high affinity and specificity for anionic phospholipids, inhibited the procoagulant activity of both SS-RBCs and (3-thalassemia RBCs, in a dose-dependent manner. More than 95% inhibition was achieved at nanomolar concentrations of annexin V. These results indicate that the procoagulant activity of both β-thalassemia RBCs and SS-RBCs may be fully ascribed to an abnormal exposure of phosphatidylserine at the outer surface of the red cells.

Nicotinamide Enhances the Polyploidization of Primary Megakaryocytes.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1366-1366
Author(s):  
Lisa M. Giammona ◽  
Eleftherios Papoutsakis ◽  
William M. Miller
Keyword(s):  
Dna Content ◽  
Molecular Mechanisms ◽  
Ex Vivo ◽  
Annexin V ◽  
Dependent Manner ◽  
Vitamin B3 ◽  
Ploidy Levels ◽  
High Ploidy

Abstract Megakaryocyte (Mk) maturation includes the development of polyploid cells via endomitosis. In vitro models of Mk differentiation can be used to gain a better understanding of the molecular mechanisms controlling this process. However, it is challenging to achieve ploidy levels in cultured human cells that are as high as those observed in vivo. Others have recently reported the use of chemical inhibitors to increase Mk ploidy (Lannutti et al., Blood 105:3875, 2005). Here, we show that nicotinamide (NIC), a form of vitamin B3, enhances the normal process of Mk polyploidization and leads to both a greater fraction of high ploidy cells and a greater degree of polyploidization. Human mobilized peripheral blood CD34+ cells were cultured in serum-free medium supplemented with thrombopoietin (TPO) to induce Mk differentiation. Beginning on day 5 of culture, cells were treated with nicotinamide (3 and 6.25 mM) and monitored for DNA content, growth, apoptosis, and surface marker expression. NIC treatment resulted in a greater fraction of Mks with high ploidy (DNA content greater than or equal to 8N). The ploidy of NIC treated cells continued to increase over the duration of the 13-day culture, whereas the ploidy of untreated cells peaked at day 9. On day 13 (8 days of NIC exposure), the percentages of high ploidy Mks for the untreated, 3 mM NIC, and 6.25 mM NIC conditions were 23%, 48%, and 63%, respectively. Furthermore, cells treated with NIC reached ploidy levels of 64N and 32N for 6.25 and 3 mM NIC, respectively, compared to 16N for untreated cells. NIC-treated cells also displayed dramatic differences in morphology - characterized by an increase in cell size, the presence of a more highly lobated nucleus, and an increased frequency of proplatelet-forming cells. Nicotinamide is known to inhibit poly(ADP-ribose) polymerase (PARP) and Sir2, which are both NAD+ dependent enzymes. Preliminary experiments show that PARP activity is low in cultured Mks and is not affected by addition of 6.25 mM NIC. Continued exposure (beginning at day 5) to the PARP inhibitors (and nicotinamide analogs) 3-aminobenzamide (3-AB) and benzamide at concentrations of 1, 3, and 6.25 mM was toxic to cells in a dose dependent manner. Interestingly, high doses of NIC (25 and 50 mM) were also toxic to cells. Remarkably, while Mk polyploidization and apoptosis are typically correlated, the increase in DNA content observed for NIC-treated cells occurred without significantly affecting the percentage of apoptotic Mks (assessed by Annexin V staining). These data suggest that it may be possible to partially decouple Mk apoptosis and polyploidization. Furthermore, while 6.25 mM NIC inhibited cell proliferation by ~35%, total expansion of cells cultured with 3 mM NIC was similar to that of untreated cells. This, combined with similar Mk commitment, as defined by a similar percentage of CD41+ cells, resulted in a greater overall number of high ploidy Mks in cultures treated with NIC. Since there is a direct correlation between Mk DNA content and platelet production (Mattia et al., Blood 99:888, 2002), these results suggest a possible therapeutic benefit of NIC for the management of thrombocytopenia. Similarly, NIC could also be used as an additive to ex vivo Mk cultures destined for transplantation. Figure Figure

Inhibition Effects of baicalin on Lymphoma Cell Line CA46 in Vitro and in Vivo

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5053-5053
Author(s):  
Jian Da Hu ◽  
Yi Huang ◽  
Yingyu Chen ◽  
Tiannan Wei ◽  
Tingbo Liu ◽  
...  
Keyword(s):  
Cell Line ◽  
Biological Effects ◽  
Annexin V ◽  
Lymphoma Cell ◽  
Control Group ◽  
Dependent Manner ◽  
Lymphoma Cell Line ◽  
Proliferation Inhibition

Abstract Baicalin is a traditional Chinese medicine with multiple biological effects. Some researches showed baicalin has anti-tumor effects in solid tumor, such as prostate cancer. In order to investigate its effects on proliferation inhibition and apoptosis induction in human lymphoma cell, we treated Burkitt lymphoma cell line CA46 with baicalin in vitro and in vivo of CA46 xenograft. Baicalin remarkably inhibited the cell proliferation, with an IC50 value of 10μM. Apoptosis was remarkably induced by baicalin in a dose-dependent manner, which was detected by Annexin V FITC/PI double staining analysis, TUNEL labeling method and DNA fragmentation respectively. Furthermore, RT-PCR showed that the mRNA expressions of c-myc and bcl-2 in treated CA46 cell decreased in a time-dependent manner. Western-Blot showed that the protein expressions of c-myc, bcl-2, procaspase-3 and PARP(116KD) in baicalin treated CA46 cell were down-regulated, while the expression of PARP(85KD) increased. Based on the results in vitro, we investigated in vivo efficacy of baicalin, alone or in combination with cytotoxic drug VP16, for treatment in CA46 nude mice xenograft. Baicalin with the dosage of 40mg/kg/d and 80kg/mg/d could remarkably inhibit the growth of the tumor compared with control group. Combination of baicalin and VP16 had better anti-tumor effects. Histological examination of tumor samples showed more necrotic cells in treated groups. And obvious apoptosis could be observed by electron microscope. No adverse events were found in treated groups. From above we could conclude that baicalin could efficiently induce proliferation inhibition and apoptosis of CA46 cells in vitro and in vivo, which may be related with the down-regulation of c-myc and bcl-2 expressions, as well as the up-regulation of caspase-3 activity.

The acceleration of the propagation phase of thrombin generation in patients with steady-state sickle cell disease is associated with circulating erythrocyte-derived microparticles

2012 ◽  
Vol 107 (06) ◽  
pp. 1044-1052 ◽  
Author(s):  
Grigoris Gerotziafas ◽  
Patrick Van Dreden ◽  
Mourad Chaari ◽  
Vassiliki Galea ◽  
Amir Khaterchi ◽  
...  
Keyword(s):  
Steady State ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Thrombin Generation ◽  
Generation Process ◽  
Cell Disease ◽  
Significant Acceleration ◽  
High Concentrations ◽  
The Impact ◽  
Functional Alteration

SummarySickle cell disease (SCD) is linked to hypercoagulability and is characterised by high concentrations of erythrocyte-derived microparticles (Ed-MPs). However, the impact of procoagulant cell-derived microparticles on the thrombin generation process remains unclear. We analysed the alterations of each phase of thrombin generation (TG) in relation to the concentration of erythrocyte- or platelet-derived microparticles (Ed-MPs and Pd-MPs) in a cohort of patients with steady-state SCD. We studied 92 steady-state SCD patients, 19 of which were under treatment with hydroxyurea, and 30 healthy age- and sex-matched individuals. TG was assessed by calibrated automated thrombogram. Ed-MP and Pd-MP expressing or not phosphatidylserine (PS) were determined by means of flow cytometry. Procoagulant phospholipid-dependent activity in the plasma was evaluated by the Procoag-PPL assay. Levels of thrombomodulin and haemoglobin in the plasma as well as red blood cell and reticulocyte counts were measured. SCD patients, independently of the administration of hydroxyurea, were marked by a significant acceleration in the propagation phase of TG which correlated with the Ed-MP/PS+ concentration. TG was significantly attenuated in hydroxyurea-treated patients. In conclusion, the acceleration of the propagation phase of TG, driven by Ed-MP/PS+, is a major functional alteration in blood coagulation in patients with steady-state SCD. Treatment with hydroxyurea, in addition to the regulation of haemolysis, lowers Ed-MPs and attenuates thrombin generation. The thrombogram could be a useful tool for the diagnosis of hypercoagulability and optimisation of the treatment in patients with SCD.

scholarly journals O-GlcNAcylation Is Essential for Rapid Pomc Expression and Cell Proliferation in Corticotropic Tumor Cells

Endocrinology ◽  
2021 ◽  
Vol 162 (12) ◽  
Author(s):  
Logan J Massman ◽  
Michael Pereckas ◽  
Nathan T Zwagerman ◽  
Stephanie Olivier-Van Stichelen
Keyword(s):  
Pituitary Adenoma ◽  
Cancer Progression ◽  
Messenger Rna ◽  
Cellular Proliferation ◽  
Endocrine Function ◽  
Dependent Manner ◽  
Cushing Disease ◽  
Human Proteins ◽  
Acth Secreting ◽  
The Impact

Abstract Pituitary adenomas have a staggering 16.7% lifetime prevalence and can be devastating in many patients because of profound endocrine and neurologic dysfunction. To date, no clear genomic or epigenomic markers correlate with their onset or severity. Herein, we investigate the impact of the O-GlcNAc posttranslational modification in their etiology. Found in more than 7000 human proteins to date, O-GlcNAcylation dynamically regulates proteins in critical signaling pathways, and its deregulation is involved in cancer progression and endocrine diseases such as diabetes. In this study, we demonstrated that O-GlcNAc enzymes were upregulated, particularly in aggressive adrenocorticotropin (ACTH)-secreting tumors, suggesting a role for O-GlcNAcylation in pituitary adenoma etiology. In addition to the demonstration that O-GlcNAcylation was essential for their proliferation, we showed that the endocrine function of pituitary adenoma is also dependent on O-GlcNAcylation. In corticotropic tumors, hypersecretion of the proopiomelanocortin (POMC)-derived hormone ACTH leads to Cushing disease, materialized by severe endocrine disruption and increased mortality. We demonstrated that Pomc messenger RNA is stabilized in an O-GlcNAc-dependent manner in response to corticotrophin-releasing hormone (CRH). By affecting Pomc mRNA splicing and stability, O-GlcNAcylation contributes to this new mechanism of fast hormonal response in corticotropes. Thus, this study stresses the essential role of O-GlcNAcylation in ACTH-secreting adenomas’ pathophysiology, including cellular proliferation and hypersecretion.

scholarly journals Inhibitory Molecules PD-1, CD73 and CD39 Are Expressed by CD8+ T Cells in a Tissue-Dependent Manner and Can Inhibit T Cell Responses to Stimulation

2021 ◽  
Vol 12 ◽  
Author(s):  
Corinne J. Smith ◽  
Christopher M. Snyder
Keyword(s):  
T Cells ◽  
Salivary Gland ◽  
T Cell ◽  
Ex Vivo ◽  
Granzyme B ◽  
Murine Cytomegalovirus ◽  
Lymphoid Tissues ◽  
Dependent Manner ◽  
Inhibitory Molecules ◽  
The Impact

The salivary gland is an important tissue for persistence and transmission of multiple viruses. Previous work showed that salivary gland tissue-resident CD8+ T cells elicited by viruses were poorly functional ex vivo. Using a model of persistent murine cytomegalovirus (MCMV) infection, we now show that CD8+ T cells in the salivary gland and other non-lymphoid tissues of mice express multiple molecules associated with T cell exhaustion including PD-1, CD73 and CD39. Strikingly however, these molecules were expressed independently of virus or antigen. Rather, PD-1-expressing T cells remained PD-1+ after migration into tissues regardless of infection, while CD73 was activated on CD8+ T cells by TGF-β signaling. Blockade of PD-L1, but not CD73, improved cytokine production by salivary gland T cells ex vivo and increased the expression of granzyme B after stimulation within the salivary gland. Nevertheless, salivary-gland localized CD8+ T cells could kill PD-L1-expressing targets in vivo, albeit with modest efficiency, and this was not improved by PD-L1 blockade. Moreover, the impact of PD-L1 blockade on granzyme B expression waned with time. In contrast, the function of kidney-localized T cells was improved by CD73 blockade, but was unaffected by PD-L1 blockade. These data show that tissue localization per se is associated with expression of inhibitory molecules that can impact T cell function, but that the functional impact of this expression is context- and tissue-dependent.

Circulating Microparticles, Plasma Protein C and Free Protein S Levels in Children with Sickle Cell Anemia.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1431-1431
Author(s):  
Andrea Piccin ◽  
Elva Eakins ◽  
Ciaran V Murphy ◽  
William G Murphy ◽  
Corrina McMahon ◽  
...  
Keyword(s):  
Steady State ◽  
Platelet Activation ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Protein C ◽  
Annexin V ◽  
Protein S ◽  
Free Protein ◽  
Sub Saharan ◽  
Lower Correlation

Abstract Microparticles (MP) originate from blebbing and shedding from cell membrane surfaces in physiological and pathological conditions. Increased levels are generated by a number of mechanisms including platelet activation, vascular endothelial damage, thrombin activity, C5b-9 activation, and PF4-heparin-antibody interaction. Increased circulating MP have been described in patients with sickle cell anemia (SCA). Elevated monocyte-derived MP expressing tissue factor have been reported in patients in crisis. The lack of a standardised method for MP quantification remains problematic. We measured MP (numbers and functional markers), protein C and free protein S, in a large cohort of pediatric patients to investigate the role of MP in SCA and their relation to protein C and free protein S plasma levels. One hundred and eleven children of sub-Saharan African ethnicity with SCA (hemoglobin (Hb) SS) were studied: 51 without previous history of crisis in steady state (mean age 5.3 years); 15 in crisis (9 chest crises, 6 other, mean age 5.4 years); 30 on hydroxyurea (mean age 7.2 years); 15 on transfusion therapy (mean age 6.5 years); 17 children of sub-Saharan African ethnicity of similar age (mean age 4.6 years) were used as control group (Hb AA). MP were analyzed by flow cytometry, according to Biró et al (J Thromb Haemost.2004; 2(10):1842–51), using Annexin V and antibodies against, CD61, CD42a, CD62P (P-selectin), CD235a, CD14, CD142 (tissue factor), CD201 (endothelial protein C receptor or EPCR), CD62E (E-selectin), CD36 (thrombospondin or TSP-1), CD47 (TSP-1 receptor), CD31 PECAM (platelet-endothelial cellular adhesion marker), CD144 (VE-cadherin). Protein C (chromogenic) and free protein S (latex based assay) were measured in all subjects. Correlation was measured by Pearson Rank test, and comparisons between groups were analyzed by Mann-Whitney test. Total MP AV were lower in crisis (1.26 × 106/ml; 0.56–2.44 × 106) and steady state (1.35 × 106/ml; 0.71– 3.0 × 106) compared to transfusion (4.33 × 106/ml; 1.6–9.2 × 106p&lt;0.01). Protein C levels were significantly lower in crisis (median 0.52 IU/ml; interquartile range 0.43–0.62) compared to all other groups: HbAA (0.72 IU/ml; 0.66–0.82, p&lt;0.001); hydroxyurea (0.67 IU/ml; 0.58–0.77, p&lt;0.001); steady state (0.63 IU/ml; 0.54–0.70, p&lt;0.05) and transfusion (0.60 IU/ml; 0.54–0.70, p&lt;0.05). In addition levels were significantly reduced in steady state (0.63 IU/ml; 0.54–0.70) compared to HbAA (0.72 IU/ml; 0.66–0.80, p&lt;0.01). Protein S levels were significantly higher in HbAA (0.85 IU/ml; 0.72–0.97) compared with crisis (0.49 IU/ml; 0.42–0.64, p&lt;0.001), hydroxyurea (0.65 IU/ml; 0.56–0.74, p&lt;0.01), and transfusion (0.59 IU/ml; 0.47–0.71, p&lt;0.01). There was also a significant difference in crisis patients compared to steady state (0.49 IU/ml; 0.42–0.64 v 0.68 IU/ml; 0.58–0.79, p&lt;0.05). There was high correlation (R&gt;0.9, p&lt;0.05) between total numbers of Annexin V positive MP (MP AV) and platelet MP expressing non-activation platelet markers. There was a lower correlation between MP AV and MP CD62P (R=0.73, p&lt;0.05) (platelet activation marker), and also a lower correlation between percentage of MP expressing CD201 (%MP CD201) and %MP CD14 (R=0.627, p&lt;0.001). %MP CD201 was higher in crisis (11.6%) compared to HbAA (3.2%, p&lt;0.05); %MP CD144 was higher in crisis (7.6%) compared to transfusion (2.1%, p&lt;0.05); %CD14 (0.77%) was higher in crisis compared to transfusion (0.0%, p&lt;0.05) and steady state (0.0%, p&lt;0.01); MP CD14 was detectable in a higher number of samples (92%) in crisis compared to the rest (40%); %MP CD235a was higher in crisis (17.9%) compared to transfusion (8.9%), hydroxurea (8.7%) and steady state (9.9%, p&lt;0.05); %CD62E did not differ significantly across the groups and CD142 was undetectable. These studies indicate that there are significantly lower levels of protein C and free protein S in children with sickle cell crisis. In addition there are significantly lower numbers of circulating platelet MP in steady state and crisis patients; however in crisis a significantly higher percentage of MP express markers of endothelial and vascular damage, and of red cell origin. Among these are composite hybrid microparticles expressing markers of more than one cell type, probably brought about by severe vascular stress and close contact of various circulating cell types with vascular endothelium.

High-Vorticity with Periodic Flow Enhances in Vitro Biogenesis of Healthy Platelets from iPSC-Derived-Megakaryocytes

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2181-2181
Author(s):  
Yukitaka Ito ◽  
Sou Nakamura ◽  
Tomohiro Shigemori ◽  
Naoshi Sugimoto ◽  
Yoshikazu Kato ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Steady State ◽  
Ex Vivo ◽  
Annexin V ◽  
Poor Quality ◽  
Flow Chamber ◽  
Physical Parameters ◽  
Platelet Production

Abstract Each transfusion requires 200-300 billion platelets in patients with thrombocytopenia. To continuously supply such a huge number of platelets by ex vivo generation, two distinct steps, megakaryopoiesis and platelet shedding, must be both considered. For the former, one approach is to increase the number of source cell, megakaryocytes. For example, the immortalized megakaryocyte cell line (imMKCL) system uses self-renewing megakaryocyte (MK) cell lines derived from induced pluripotent stem cells (iPSCs) (Nakamura et al., Cell Stem Cell, 2014). For the latter, there have been an idea of bioreactors whereby shedding of platelets from proplatelets could be promoted by flow-dependent shear force within the bone marrow in vivo (Junt et al., Science, 2007; Zhang et al., J Exp Med, 2012). Based upon this idea, we constructed a flow chamber type bioreactor recapitulating in vivo blood flow shear rate. However, this bioreactor failed to efficiently yield platelets, and moreover, the produced platelets had poor quality as indicated by high Annexin V levels (Exp Hematol, 2011 and unpublished result). Recently, we demonstrated two different kinetics of platelet biogenesis from bone marrow MKs, whereby either thrombopoietin (TPO) mostly regulates steady-state shedding of platelets from proplatelets, or interleukin-a (IL-1a) triggers inflammation-dependent rupture of MK cytoplasm contributing to a quick increase of platelet count at higher rate (Nishimura et al., J Cell Biol, 2015). However, the rupture type platelets revealed shorter half-life with relatively higher Annexin V levels. Therefore, to gain insights from platelet biogenesis in vivo, we focused on biophysical analysis of steady-state platelet biogenesis via proplatelets in bone marrow. Our observations strongly indicated that the presence of 'vorticity' defined by vortex turbulence in addition to shear-dependent 'stress' and 'strain' correlates with the efficient shedding of competent platelets. From this new finding, we developed an alternative bioreactor system, which enabled generation of 100 billion platelets from imMKCL in a 16L-scale liquid culture condition without any adherent machinery using two 10L-bioreactors. Furthermore, platelets generated via new bioreactors showed low Annexin V levels (<10-15%) and shortened bleeding time post transfusion into NOG mice and rabbits with thrombocytopenia, comparable to human blood product platelets. Regarding the platelet production using WAVE bag system (GE healthcare, UK), the system is already clinically available for cord blood cell expansion in most countries, but lacks adequate levels of vorticity and shear strain/stress. Accordingly, the produced platelets had high Annexin V levels (i.e., 50-65%) as well as diminished yield efficiency (P<0.001). In conclusion, our study has uncovered the novel biophysical aspect of platelet biogenesis. The application of the new set of physical parameters in constructing large sized bioreactors shall facilitate the industrialization of platelet production. Disclosures Eto: Megakaryon Co. Ltd.: Research Funding.

Relationship between Autophagy and Ventilator-induced Diaphragmatic Dysfunction

2015 ◽  
Vol 122 (6) ◽  
pp. 1349-1361 ◽  
Author(s):  
Ilan Azuelos ◽  
Boris Jung ◽  
Martin Picard ◽  
Feng Liang ◽  
Tong Li ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Messenger Rna ◽  
Ex Vivo ◽  
Diaphragm Muscle ◽  
Autophagosome Formation ◽  
Diaphragmatic Dysfunction ◽  
Transmission Electron ◽  
Diaphragmatic Weakness ◽  
The Impact

Abstract Background: Mechanical ventilation (MV) is associated with atrophy and weakness of the diaphragm muscle, a condition termed ventilator-induced diaphragmatic dysfunction (VIDD). Autophagy is a lysosomally mediated proteolytic process that can be activated by oxidative stress, which has the potential to either mitigate or exacerbate VIDD. The primary goals of this study were to (1) determine the effects of MV on autophagy in the diaphragm and (2) evaluate the impact of antioxidant therapy on autophagy induction and MV-induced diaphragmatic weakness. Methods: Mice were assigned to control (CTRL), MV (for 6 h), MV + N-acetylcysteine, MV + rapamycin, and prolonged (48 h) fasting groups. Autophagy was monitored by quantifying (1) autophagic vesicles by transmission electron microscopy, (2) messenger RNA levels of autophagy-related genes, and (3) the autophagosome marker protein LC3B-II, with and without administration of colchicine to calculate the indices of relative autophagosome formation and degradation. Force production by mouse diaphragms was determined ex vivo. Results: Diaphragms exhibited a 2.2-fold (95% CI, 1.8 to 2.5) increase in autophagic vesicles visualized by transmission electron microscopy relative to CTRL after 6 h of MV (n = 5 per group). The autophagosome formation index increased in the diaphragm alone (1.5-fold; 95% CI, 1.3 to 1.8; n = 8 per group) during MV, whereas prolonged fasting induced autophagosome formation in both the diaphragm (2.5-fold; 95% CI, 2.2 to 2.8) and the limb muscle (4.1-fold; 95% CI, 1.8 to 6.5). The antioxidant N-acetylcysteine further augmented the autophagosome formation in the diaphragm during MV (1.4-fold; 95% CI, 1.2 to 1.5; n = 8 per group) and prevented MV-induced diaphragmatic weakness. Treatment with the autophagy-inducing agent rapamycin also largely prevented the diaphragmatic force loss associated with MV (n = 6 per group). Conclusions: In this model of VIDD, autophagy is induced by MV but is not responsible for diaphragmatic weakness. The authors propose that autophagy may instead be a beneficial adaptive response that can potentially be exploited for therapy of VIDD.

Malignant transformation in melanocytes is associated with increased production of procoagulant microvesicles

2011 ◽  
Vol 106 (10) ◽  
pp. 712-723 ◽  
Author(s):  
Luize Lima ◽  
Andreia Oliveira ◽  
Luiza Campos ◽  
Martin Bonamino ◽  
Roger Chammas ◽  
...  
Keyword(s):  
Malignant Transformation ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Biological Effects ◽  
Flow Cytometric Analysis ◽  
Dependent Manner ◽  
Initiator Protein ◽  
Prothrombinase Complex ◽  
Thrombosis Model

SummaryShedding of microvesicles (MVs) by cancer cells is implicated in a variety of biological effects, including the establishment of cancer-associated hypercoagulable states. However, the mechanisms underlying malignant transformation and the acquisition of procoagulant properties by tumour-derived MVs are poorly understood. Here we investigated the procoagulant and prothrombotic properties of MVs produced by a melanocyte-derived cell line (melan-a) as compared to its tumourigenic melanoma counterpart Tm1. Tumour cells exhibit a two-fold higher rate of MVs production as compared to melan-a. Melanoma MVs display greater procoagulant activity and elevated levels of the clotting initiator protein tissue factor (TF). On the other hand, tumour- and melanocyte- derived MVs expose similar levels of the procoagulant lipid phosphatidylserine, displaying identical abilities to support thrombin generation by the prothrombinase complex. By using an arterial thrombosis model, we observed that melanoma- but not melanocyte-derived MVs strongly accelerate thrombus formation in a TF-dependent manner, and accumulate at the site of vascular injury. Analysis of plasma obtained from melanoma-bearing mice showed the presence of MVs with a similar procoagulant pattern as compared to Tm1 MVs produced in vitro. Remarkably, flow-cytometric analysis demonstrated that 60% of ex vivo MVs are TF-positive and carry the melanoma-associated antigen, demonstrating its tumour origin. Altogether our data suggest that malignant transformation in melanocytes increases the production of procoagulant MVs, which may contribute for a variety of coagulation- related protumoural responses.

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