Desialylation of Megakaryocytes Diminishes Platelet Production By Disrupting Megakaryocyte Adhesion, Migration and Proplatelet Formation in Chronic Immune Thrombocytopenia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 165-165
Author(s):  
Qian-Ming Wang ◽  
Jia-Min Zhang ◽  
Xiao-Lu Zhu ◽  
Hai-Xia Fu ◽  
Meng Lv ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Immune Thrombocytopenia ◽  
Stromal Cell ◽  
Bleeding Tendency ◽  
Hematopoietic Stem ◽  
Platelet Production ◽  
Vascular Niche ◽  
Bm Niche ◽  
Sialidase Inhibitor ◽  
Proplatelet Formation

Abstract Immune thrombocytopenia (ITP) is a common autoimmune disorder characterized by increased bleeding tendency and isolated thrombocytopenia. The precise pathogenesis of the decreased thrombopoiesis in chronic ITP (cITP) is poorly understood. Megakaryocytes (MKs) in cITP show impaired maturation and thrombopoiesis, which are correlated with numerous glycoproteins on the surface of MKs. Different types of sialoglycoproteins are expressed on the surface of megakaryocytes, including GPIbα and platelet endothelial cell adhesion molecule-1 (PECAM-1), both of which participate in megakaryocyte migration to the vascular niche in the bone marrow (BM) and in proplatelet formation. Desialylation has recently been identified a contributor to the pathogenesis of thrombocytopenia. Our previous study has demonstrated that desialylation of GPIbα is related to increased apoptosis and phagocytosis of platelets in cases of prolonged isolated thrombocytopenia after allogeneic hematopoietic stem cell transplantation (Zhang et al., J Hematol Oncol, 2015). Because MKs are heavily sialylated cells, we raised the question whether the desialylation of megakaryocytes contributes to the defective thrombopoiesis in patients with cITP through impaired MK migration, adhesion and proplatelet formation in the vascular niche. MK desialylation was analyzed by flow cytometry using lectins. Desialylated glycoproteins were measured using selective exo-enzymatic labeling. Protein expression, distribution and interaction were measured using the following techniques: immunofluorescence, flow cytometry, western blot and immunoprecipitation. cITP MKs exhibited increased β-galactose exposure compared to the control MKs, indicating excessive desialylation. Desialylation was correlated with decreased platelet production of MKs. We further explored the cause of desialylation and found that the sialidase NEU1 was over-expressed in MKs. Treatment with the sialidase inhibitor DANA ameliorated the loss of sialic acids. These results indicated that NEU1 contributed to the desialylation of MKs in cITP. Altered MK distribution in the BM niche was exhibited upon BM biopsy of cITP patients. The ratio of perivascular MKs was markedly decreased in cITP patients. Defective adhesion and transmigration behaviors were also discovered in desialylated cITP MKs. The motility of cITP MKs through stromal cell monolayers driven by stromal cell derived factor 1 (SDF1) was decreased. Adherence to fibronectin, collagen and fibrinogen was assessed, and desialylated cITP MKs exhibited an increase in adhesion with these macromolecules. Similar abnormalities were observed in the BM niche of ST6Gal1-/- mice, and treatment with ST6Gal1 and CMP-SA augmented the ratio of MKs in the BM vascular niche in ST6Gal1-/-mice, indicating that desialylation impaired the MK migration and adhesion. Additional experiments focused on which specific sialoglycoproteins are excessively desialylated. As detected by SEEL, PECAM-1 exhibited excessive desialylation in cITP MKs, which was related to impaired CXCR4 polarization in response to SDF1. Inhibition of sialidase using DANA partially restored this polarization, demonstrating that desialylation of PECAM-1 was responsible for the defect in MK migration to the vascular niche. Meanwhile, PECAM-1 desialylation was associated with GPIIb/IIIa overactivation, which correlated to the increased adhesion of MKs. This increased adhesion was reversed by the GPIIb/IIIa inhibitor lotrafiban, indicating that desialylated PECAM-1 contributed to the abnormal adhesion via overactivation of GPIIb/IIIa. Desialylation of GPIbα was found on the surface of MKs from cITP patients and was associated with abnormal microtubule formation and increased MK apoptosis through altered 14-3-3ζ distribution, which led to the impediment of proplatelet formation in the vascular niche. In conclusion, our results demonstrate that MKs are desialylated by NEU1 in cITP patients, and desialylated MKs present with defective migration towards the vascular niche, abnormal adhesion to the extracellular matrix and impaired proplatelet formation. Desialylation of PECAM-1 and GPIbα have been demonstrated to be responsible for these abnormal behaviors of MKs. Sialidase inhibitor shows an improvement in the thrombopoiesis of cITP MKs; therefore, our study implies a novel potential approach for the treatment of cITP. Disclosures No relevant conflicts of interest to declare.

Vascular Niche and Platelet Production: Stromal Cells Inhibit Proplatelet and Platelet Formation by Human Megakaryocytes (MK), Whereas Endothelial Cells Are Permissive

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 402-402
Author(s):  
Yasmine Ouzegdouh ◽  
Laurence Momeux ◽  
Elisabeth Cramer-Borde
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Endothelial Cells ◽  
Growth Factors ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Cell Types ◽  
Platelet Production ◽  
Proplatelet Formation ◽  
Platelet Formation

Abstract Abstract 402 The site of platelet production in the human body is still unclear, but several evidence, including the recent demonstration by our team that shear stress in vitro accelerates platelet formation, favour the hypothesis of an intravascular location of the platelet release process. Therefore we have undertaken the following study to compare the effect of two cell types from the human bone marrow microenvironment, stromal medullary cells and endothelial cells, on the final steps of megakaryocyte (MK) maturation and platelet production. Firstly, supporting the goal of our study, we show that entire mature MK can be encountered in the lumen of normal bone marrow sinusoids and therefore enter the circulation of flowing blood; Secondly, we have used a new microcapillary device coated with von Willebrand factor and were able to confirm by videomicroscopy that exposure of human mature MK to shear forces accelerates proplatelet extension and facilitates platelet liberation from the tips of proplatelets. During this process, the nuclear lobes of polyploid MK tend to separate and to form distinct proplatelet units ready to deliver platelets: this observation might explain why massive senescent MK nuclei are only rarely found in human tissues, bone marrow or lung. Then, we used flow cytometry, optical and electron microscopy, to document and visualise the interaction of human medullary stromal cells (HS5), murine medullary stromal cells (MS5), and human endothelial cells (HUVEC) with the final steps of human MK maturation, namely proplatelet formation. MK were grown from umbilical cord blood CD34+ cells in the presence of Stem Cell Factor (SCF) and thrombopoietin and co-cultured with stromal cells between day 10 and day 13 of culture. Stromal cells virtually completely inhibited proplatelet formation from MK, whereas endothelial cells showed no effect and allowed the emission of proplatelets. EM and flow cytometry confirmed that stromal cells blocked proplatelet and platelet formation and showed that this was accompanied with a considerable development of demarcation membranes which coincided with a 60% increase of CD41 expression by MK. In parallel, stromal cells induced a reduction of apoptosis signs with a reduction of annexin V fixation by maturing MK. Since the step of proplatelet formation is accompanied with cytoplasmic apoptotic signs, among which increased phosphatidyl serine exposure, this result is in accordance with the observed blockade of proplatelet extension. Stromal cell secretion include several growth factors, namely SCF and GMCSF. MK were cultured in the presence of stromal cell culture supernatants, and with these growth factors either individually or mixed together, but this had no effects on proplatelet production, suggesting that physical contacts between the two cell types are necessary. In conclusion, our data show that human bone marrow microenvironment (stromal cells) has an inhibitory effect on proplatelet and platelet formation whereas the vascular microenvironment (endothelial cells) is permissive. This may explain the absence of proplatelets in the bone marrow parenchyma and be an additional evidence that platelet formation and release has to mainly occur in the intravascular compartment. Disclosures: No relevant conflicts of interest to declare.

Abnormalities of Bone Marrow Immune Micro-Environment in Patients with Immune Thrombocytopenia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3464-3464
Author(s):  
Yang Song ◽  
Yu-tong Wang ◽  
Xiao-jun Huang ◽  
Yuan Kong
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
Endothelial Cells ◽  
Th17 Cells ◽  
Immune Thrombocytopenia ◽  
Perivascular Cells ◽  
Platelet Production ◽  
Cd34 Cells ◽  
Significant Difference

Abstract Background: Immune thrombocytopenia (ITP) is an immune-mediated disease that is characterized by excessive platelet destruction and decreased platelet production. Although antiplatelet antibodies are considered as the primary immunologic defect in ITP, dysfunctional cellular immunity is also important in the pathophysiology of ITP. The current publications have observed excessive activation and proliferation of platelet auto-antigen-reactive CTLs, production abnormal Th cells, abnormal numbers and function of Tregs in peripheral blood of ITP, but no one focus on the bone marrow (BM) micro-environment in ITP patients. Many cell types including osteoblastic, perivascular, endothelial cells, and various mature immune cells contribute to the BM micro-environment. We have recently reported that the impaired BM vascular micro-environment may affect the thrombopoiesis of CD34+ cells by disrupting the interaction between megakaryocytes and BM endothelial cells (BMECs), resulting in the delayed platelet engraftment in allotransplant patients with prolonged isolated thrombocytopenia (Kong Y, et al. Biol Blood Marrow Transplant. 2014; 20:1190-1197). In mice model, the cross-talk between megakaryocytes and BMECs in BM vascular micro-environment regulates the megakaryocyte maturation and thrombopoiesis. Therefore, we hypothesized that the abnormal BM vascular micro-environment and immune micro-environment may operate in the occurrence of ITP. Aims: To investigate whether abnormal BM vascular and immune micro-environment are involved in ITP patients. Methods: The compartments of BM immune micro-environment were analyzed by flow cytometry in 26 untreated ITP patients and 26 healthy donors (HD). The fractions of T cells, including Th1, Tc1,Th2, Tc2 ,Th17 and Treg were identified as CD3+ CD8- IFN-gama+, CD3+ CD8- IFN-gama+, CD3+ CD8+ IL4+, CD3+ CD8+ IL-4+, CD3+ CD8- IL17A+ and CD3+ CD4+ CD25+ Foxp3+, respectively. The BMECs and perivascular cells, acting as key elements of vascular micro-environment, were identified as CD45- CD34+ VEGFR2+ and CD45- CD34- CD146+, respectively. Hematoxylin-eosin (H&E) staining and immunohistochemistry (IHC) using rabbit anti-human CD34 and CD146 primary antibodies were performed on each BM trephine biopsies (BMB) derived from the patients and controls. Results: The proportion of Th1 cells and Tc1 cells among the bone marrow mononuclear cells (BMMNCs) was significantly increased in ITP patients compared to HD (27.7% ± 11.6% vs. 16.3% ± 7.7%, P<0.001; 39.8%±17.7% vs. 24.1%±11.8%, P<0.005), whereas there was no significant difference in the percentages of Th2 and Tc2 cells. In addition, the proportion of Th17 cells in ITP patients was remarkable higher than HD (3.2%±0.51%1.5%vs 1.7%±1.0%, P<0.0001). We also found the significantly decreased percentage of Treg in ITP patients compared to HD (2.5%±2.0% vs 3.7%±2.6%, P<0.001). However, the frequency of CD34+ cells as well as BMECs and perivascular cells were similar in BM between the ITP patients and HD. Consistent with our flow cytometry data, histological analysis of the recipient BMBs in situ showed no significant differences in CD34-positive BMECs and CD146-positive perivascular cells between ITP patients and HD. Summary/Conclusion: The BM CD34+ cells and vascular micro-environment were normal in ITP patients. However, the abnormal BM immune micro-environment, including the excessive polarization of Th1, Tc1 and Th17 cells and a remarkable decrease of Treg cells were observed in ITP patients. Our data indicated that the desregulated T cells responses in BM may abrogate the thrombopoiesis through the impaired megakaryocytes maturation and decreased platelet production, and eventually contributing to the occurrence of ITP. Acknowledgment: Supported by the National Natural Science Foundation of China (grant nos. 81370638&81230013), and the Beijing Municipal Science and Technology Program (grant nos. Z141100000214011& Z151100004015164& Z151100001615020). Disclosures No relevant conflicts of interest to declare.

Development Of 3D Models For Studying Megakaryopoiesis In Vitro

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3696-3696
Author(s):  
Lindsay Wray ◽  
Christian A Di Buduo ◽  
David L. Kaplan ◽  
Alessandra Balduini
Keyword(s):  
Mechanical Properties ◽  
Bone Marrow ◽  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Peripheral Blood ◽  
Platelet Production ◽  
Vascular Niche ◽  
Proplatelet Formation ◽  
Bone Marrow Vascular Niche

Abstract Introduction Silk fibroin, derived from Bombyx mori silkworm cocoons, is used extensively in biomaterials and regenerative medicine. The useful characteristics of this protein include self-assembly, robust mechanical properties, biocompatibility and biodegradability. Moreover, silk can be enhanced through a variety of chemical modifications that affect cell attachment, growth and differentiation. Thrombocytopenia occurs when a patient suffers from an abnormally low platelet count in the peripheral blood; usually a result of disease, trauma, or cancer treatment. To treat these patients, it is estimated that two million platelet transfusions are performed in the U.S. each year. This high demand for platelets has created a clinical demand for studying the causes of thrombocytopenia and alternative routes for treatment. Platelets are anuclear cells that are released into the bloodstream in the bone marrow by megakaryocytes via the extension of long filaments called proplatelets. It is hypothesized that platelet production from megakaryocytes is regulated by environmental factors at the site of bone marrow vascular niche. Studies of megakaryopoiesis are typically performed on extracellular matrix protein-coated culture plates and transwell membranes. While these initial studies have provided invaluable insight into the process of megakaryopoiesis, the goal of the present project was to create a bone marrow model that mimics the vascular niche for functional in vitro platelet production. We hypothesized that a silk-based in vitro tissue model would allow the effects of substrate surface properties and endothelial co-culture on megakaryopoiesis to be studied in a holistic manner, thereby enabling further elucidation of the mechanisms involved in the process of platelet production. Results In order to more closely mimic the bone marrow vascular niche structure, a porous silk sponge was assembled around the silk vessel-like tubes. Megakarycytes seeded in the porous silk sponge migrated toward the silk tube and released platelets into the tube lumen. The perfusion bioreactor moved the platelets into the platelet collecting bags. After perfusion the platelets were collected and analyzed by flow cytometry. The bioreactor platelets exhibited similar morphology, CD41 positive staining, and activation compared to peripheral blood platelet controls. Megakaryocyte attachment and proplatelet formation through the silk vascular wall were improved by altering the silk properties. Silk functionalized by entrapping extracellular matrix proteins within the tube membrane resulted in increased megakaryocyte attachment and proplatelet compared to unfunctionalized silk tube controls. Silk surface roughness improved megakaryocyte attachment compared to the control but did not affect proplatelets. Decreasing the silk stiffness improved proplatelets, but did not significantly affect megakaryocyte attachment. Co-culture with endothelial cells improved megakaryocyte attachment while maintaining a high level of proplatelet formation. Additionally, megakaryocyte and endothelial cell co-culture on the silk vessel model resulted in an icreased platelet production compared to megakaryocytes cultured alone. Conclusions The goal of this project was to develop an in vitro model of megakaryopoiesis using a tissue engineering approach. Using human megakaryocytes and endothelial cells, we demonstrate the following advanced features of the silk-based model: (1) immobilization of extracellular matrix components within the membrane, (2) tunable surface topography, (3) tunable mechanical properties, (4) physiologically relevant thickness for appropriate proplatelet extension, and (5) controlled localization of a vascular endothelium. Thus, by functionalizing silk, we can control megakaryocyte function on silk. The broader impact of this work offers a versatile new tool for studying megakaryocyte development and platelet production in vitro. Disclosures: No relevant conflicts of interest to declare.

C-MYC and NF-E2 Genes Regulate Proplatelet Formation in Cultured Megakaryocytes with Different Levels of Polyploidization.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2289-2289
Author(s):  
Mauro P. Avanzi ◽  
Jennifer G. Davila ◽  
Francine Goldberg ◽  
W. Beau Mitchell
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Wide Spectrum ◽  
The Other ◽  
Transfusion Therapy ◽  
Hematopoietic Stem ◽  
Platelet Production ◽  
Proplatelet Formation

Abstract Abstract 2289 Introduction: One of the goals of stem cell medicine is the production of platelets from stem cells for transfusion therapy. The process of platelet production from megakaryocytes is complex and depends on a wide spectrum of internal and external stimuli. The degree of cell polyploidization, amount of demarcation membrane system (DMS), and the cells capacity to form proplatelets are major determines of the quantity of platelets released by each megakaryocyte. Both polyploidization and proplatelet formation depend on the synchronous function of microtubules and actin/myosin. This complex machinery is regulated in part by the actions of the C-Myc and NF-E2 promoters. These promoters play a crucial role during polyploidization and proplatelet formation. We have examined the potential role of these promoters in the efficacy of various chemical and cell culture-based methods of driving hematopoietic stem cells to megakaryocyte differentiation and platelet production. Methods: Human cord blood derived CD34+ cells were isolated and 5×104cells were cultured with thrombopoietin (TPO) and stem cells factor (SCF) for 12 days. Megakaryocytes were cultured along with reagents that inhibit distinct mechanisms of the cytokinesis process: Rho-Rock inhibitor, Y27632 (RRI); Src-inhibitor, SU6656 (SI); Nicotinamide (NIC); Aurora-B inhibitor, ZM447439 (ABI); and Myosin Light Chain Kinase Inhibitor (MLCKI). Combinations of reagents were used in order to determine their interactions and to maximize megakaryocyte ploidy. The DMS was analyzed and quantified with Di-8 ANEPPS in flow cytometry and morphology was studied with Electron Microscopy (EM). On day 12 proplatelets were analyzed with an inverted microscope and platelets were counted with an Advia 120 cell counter. Total RNA was extracted and analyzed for C-Myc and NF-E2 mRNA by QRT-PCR. Results: All treatments increased megakaryocyte ploidy, except MLCKI. RRI reached the highest ploidy (p=0.0007), followed by NIC (p=0.003), SI (p=0.026) and ABI (p=0.018). Combinations all significantly increased polyploidization; however the only combination that equaled RRI alone was the combination of all of the other inhibitors (p<0.0001). EM showed normal megakaryocyte structure. DMS quantification showed that higher ploidy megakaryocytes had more extensive DMS (p<0.02). Higher ploidy megakaryocytes released more proplatelets and platelets than control and low ploidy cells (p=0.01). Treatments that had the lowest increase proplatelet formation were ABI and the combination of NIC-SI-ABI-MLCKI. RRI-NIC resulted in the highest release of proplatelet formation. C-Myc gene expression was down-regulated in most of the treatments. NF-E2 expression was up-regulated in megakaryocytes treated with ABI and in the combination of NIC-SI-ABI-MLCKI, but not in other treatments. Conclusion: RRI proved to be the most effective agent in driving megakaryocyte polyploidization. The summation of effects of all of the other cytokinesis inhibitors increased polyploidization only to the same extent as RRI. Light and EM imaging showed that the cultured megakaryocytes were morphologically normal. Higher ploidy megakaryocytes with an extended DMS were able to extend more proplatelets and release more platelets in culture, especially after treatment with RRI-NIC. Gene expression analysis showed that down-regulation of C-Myc in late stages of development was correlated with increased proplatelet formation and platelet release. NF-E2 up-regulation on day 12 was associated with ABI treatment and decreased proplatelet formation in cultured megakaryocytes. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Evaluation of Platelet Function Defects in Patients with Immune Thrombocytopenia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1021-1021
Author(s):  
Elena Monzón Manzano ◽  
María Teresa Alvarez Román ◽  
Andres Ramirez Lopez ◽  
Elena G Arias-Salgado ◽  
Paula Acuña ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Sialic Acid ◽  
Platelet Function ◽  
Research Funding ◽  
Immune Thrombocytopenia ◽  
Healthy Controls ◽  
Activation Markers ◽  
Platelet Production ◽  
Neuraminidase Activity ◽  
Fibrinogen Receptor

Abstract Background: Primary immune thrombocytopenia (ITP) is a megakaryocytic (MK)/platelet-specific autoimmune disorder characterized by platelet count &lt;100×10 9/L with or without bleeding manifestations, and diagnosed by exclusion of other causes of thrombocytopenia. It is widely accepted the involvement of platelet autoantibodies on deterioration of platelets from patients with ITP. Moreover, an enhanced activity of neuraminidase may also reduce sialic acid from glycoside residues on platelet surface, especially from the highly glycosylated von Willebrand factor (vWF) receptor. Because controversial results regarding the functionality of platelets from ITP patients can be found in literature, we aimed to determine platelet ability to be stimulated by agonists. Moreover, we aimed to determine the way anti-platelet auto- antibodies (abs) and neuraminidase activity may affect the function of platelets derived from MKs of healthy controls. Methods: This observational, prospective and transversal study included 42 patients with chronic primary ITP and 55 healthy controls. Platelet fibrinogen and vWF receptors and activation markers (PAC1 binding to activated fibrinogen receptor and exposure of P-selectin after agonists treatment), were evaluated by flow cytometry. Presence of Antibodies (abs) against platelet's glycoproteins in ITP serum was analysed with a Luminex based assay (LifecodesPak Lx). Neuraminidase (NEU) activity in serum was determined with the substrate 20-(4-methylumbelliferyl)-a-D-N-(MUNANA). Human CD34 + cell-enriched population was obtained with CliniMACS (MiltenyiBiotec) from G-CSF mobilized peripheral blood of a healthy donor. For MK differentiation, CD34 + cells were cultured 12 days in StemSpan™ Serum-Free Expansion Medium II (SFEM II) with 50ng/ml of recombinant human thrompoietin. Then, 10% of serum from healthy controls (4) or ITP patients (4) were added to the culture of mature MKs and incubated for 3 days. Phenotypic analysis of MKs and culture derived-platelets was carried out using abs against CD34, CD41, CD42a and CD42b.Platelet-like particles were considered as CD41-positive events with a size (FSC) and granularity (SSC) scatter properties similar to blood platelets. Culture-derived platelets were stimulated with 100 µM TRAP and 10 µM ADP and activation markers were analyzed by flow cytometry. Results: Expression of fibrinogen receptor on platelets from ITP patients were similar to those from healthy controls but showed a reduced capacity to be activated. Impairment in platelet degranulation measured as exposition of P-selectin after agonist's stimulation was also observed in platelets from these patients (Figure 1). Of note, surface content of CD42b subunit of vWF receptor was reduced (Figure 1). To determine whether diminished platelet function might be due to a plasma component, we induced platelet production from MK of healthy controls as referred in Methods. Abs against platelets and neuraminidase activity were determined in serum samples. Serum from 4 healthy controls or from 4 ITP patients (1 with anti-CD42b, 1 with anti-GPIa-IIa and 2 with undetectable abs) were added to MKs culture. No differences existed in MK differentiation and platelet production between MKs incubated with serum from healthy controls or from ITP patients, but similarly as observed in platelets from ITP patients, MK-derived platelets had an impaired ability to be activated (Table 1). Platelets derived from MKs incubated with ITP serum with anti-platelet abs had also a diminished exposure of CD42b (73±8% of controls). Moreover, neuraminidase content of these samples was slightly higher than that from ITP samples without abs (130 vs 100 % of controls). Conclusion: Platelets from ITP patients had a diminished ability to be stimulated. In vitro study showed that megakaryopoiesis was normal in presence of ITP serum, but released platelets had a lower ability to be activated. Involvement of abs in this effect cannot be ruled out despite we detected abs only in 2 of the tested sera because efficiency of method to detect these abs is ~ 50%. On the other hand, reduced levels of CD42b might be due to the increased activity of neuraminidase. Reduction of sialic acid from CD42b might initiate its metalloproteinase-mediated cleavage or change affinity of the ab used for its detection. Research funded by ISCIII-Fondos FEDER PI19/00772 and Platelet Disorder Support Association Figure 1 Figure 1. Disclosures Alvarez Román: Pfizer: Consultancy, Honoraria, Research Funding; Octapharma: Consultancy, Honoraria, Research Funding; Sobi: Consultancy, Honoraria, Research Funding; Grifols: Consultancy, Honoraria, Research Funding; Biomarin: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; CSL-Behring: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Novo-Nordisk: Consultancy, Honoraria, Research Funding. García Barcenilla: Roche: Speakers Bureau; Takeda: Speakers Bureau; Bayer: Speakers Bureau; SOBI: Speakers Bureau. Canales: Janssen: Consultancy, Honoraria, Speakers Bureau; Celgene/Bristol-Myers Squibb: Consultancy, Honoraria; Gilead/Kite: Consultancy, Honoraria; Eusa Pharma: Consultancy, Honoraria; Incyte: Consultancy; Karyopharm: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Sanofi: Consultancy; iQone: Honoraria; Sandoz: Honoraria, Speakers Bureau; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Speakers Bureau; Takeda: Consultancy, Honoraria, Speakers Bureau. Jiménez-Yuste: Grifols: Consultancy, Honoraria, Research Funding; NovoNordisk: Consultancy, Honoraria, Research Funding; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; CSL Behring: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; BioMarin: Consultancy; Sobi: Consultancy, Honoraria, Research Funding; Octapharma: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding. Butta: Novo-Nordisk: Speakers Bureau; Takeda: Research Funding, Speakers Bureau; Roche: Speakers Bureau; CSL-Behring: Research Funding.

In Vivo Platelet Production Occurs from an Expanding Pool of Non-Apoptotic Megakaryocytes in a Mouse Model of Passive Immune Thrombocytopenia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1214-1214
Author(s):  
Harald Schulze ◽  
Silke Schwiebert ◽  
Kathrin Roth ◽  
Oliver Meyer ◽  
Gabriele Strauss ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Immune Thrombocytopenia ◽  
De Novo ◽  
Hematopoietic Stem ◽  
Platelet Production ◽  
Platelet Antibodies

Abstract Abstract 1214 Immune thrombocytopenia (ITP) is the most common acquired thrombocytopenia in children. Typically, external triggers as infections or vaccinations cause the rise of antibodies that crossreact with antigens expressed on the platelet surface. These anti-platelet antibodies are mostly directed against glycoprotein complexes GPIIb/IIIa or GPIb/IX/V, resulting in an increased turnover of antibody-decorated platelets which are then sequestered by the reticuloendothelial system. Recently, it has been suggested that thrombocytopenia might also be due to an insufficient platelet production as serum of some patients with ITP can impair the maturation of CD34+ hematopoietic stem cells to bone marrow megakaryocytes (MKs) in vitro or abrogate the formation of proplatelets in an in vitro culture system. The accelerated platelet turnover demands the generation of platelets de novo. Bone marrow smears often reveal normal or slightly increased MKs, although they seem to be smaller and of altered morphology. However, very little is known about the consequences of anti-platelet antibodies on bone marrow MKs in vivo and in situ. Here, we took advantage of a simple animal model of passive ITP by single or multiple intraperitoneal injections of an anti-GPIb antibody into mice. MKs were evaluated by multi-color immunofluorescence histology on whole femur sections in a modified staining procedure that bypasses decalcification. MK numbers on day 3 were doubled in response to a single injection and tripled on day 8 when mice were injected additionally on day 3 and 7. In these mice platelet counts were up to 2000/nL on day 10, indicating the power to produce platelets. MK area per section was transiently upregulated on day 3 in single injected mice and quadrupled after multiple injections on day 8 before shrinking below norm on day 14. Staining with an anti-rat IgG antibody showed that the antibody was present on MKs within the bone marrow several hours to days after injection. The signal was present for 5 days and no antibody was detected on day 7. MKs had an overall normal morphology and showed no signs of apoptosis or DNA blebbing. All MKs analyzed were negative for TdT in a classical TUNEL assay, indicating that there were no single strand breaks. As platelet counts rose markedly while the antibody was still present on the MK surface, we sought to identify whether the pool of MKs is expanded or formed de novo. To address this, mice where fed with nucleotide analogue EdU for up to 12 days and femur sections stained with Click-It-647 reagent to stain for newly incorporated DNA while mice were treated with anti-platelet antibody or isotype control. We found EdU-positive MKs after 12 days in control isotype-injected mice indicating the de novo formation from hematopoietic stem cells. In antibody-injected mice, newly formed MKs were negative or stained weakly for EdU on day 12, suggesting that they arise partially from an existing pool of progenitors. Finally, we analyzed platelet formation in vivo by imaging of the cranial bone marrow of GPIIb-eYFP-heterozygous mice. The depletion antibody was labeled with Atto-590-fluorophore and injected hours before imaging. Vasculature was counterstained by Quantum dots. We found that MKs residing at the bone marrow were decorated with the antibody and released pre- and proplatelets into the vasculature, indicating that platelet biogenesis can occur in the presence of anti-platelet antibodies on MKs. Our data thus provide novel insight into the pathomechanism of platelet production in patients with ITP. Disclosures: No relevant conflicts of interest to declare.

Spatial and Temporal Fluctuations In Marrow SDF-1 Following Radiation Injury Regulate Megakaryocyte-Vascular Niche Interactions and Circulating Platelet Levels

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 568-568 ◽  
Author(s):  
Lisa M Niswander ◽  
Katherine H Fegan ◽  
Paul D Kingsley ◽  
Kathleen E McGrath ◽  
James Palis
Keyword(s):  
Bone Marrow ◽  
Spatial Distribution ◽  
Conflicts Of Interest ◽  
Therapeutic Interventions ◽  
Post Injury ◽  
Hematopoietic Stem ◽  
Platelet Production ◽  
Vascular Niche ◽  
Endosteal Niche

Abstract The development of megakaryocytes (MKs) in the bone marrow progresses spatially from the endosteal niche, which promotes MK progenitor proliferation, to the sinusoidal vascular niche, the site of terminal maturation and thrombopoiesis. The chemokine SDF-1 (CXCL12), signaling through receptor CXCR4, is produced by stromal cell populations throughout the marrow and is implicated in the maturational chemotaxis of MKs to the sinusoids. Understanding the regulation of MK localization has significance not only for optimal platelet production and the development of therapies for thrombocytopenia, but also in light of the recently proposed role for MKs in supporting hematopoietic stem cells (Heazlewood et al. 2013). In the injury setting of lethal total body irradiation (TBI), it was observed that radioresistant mature MKs relocate to the endosteal niche (Dominici et al. 2009, Olson et al. 2013). Complicating the study of marrow niches post-TBI is the vascular dilation that accompanies the drastic loss of marrow cells. Having confirmed that MKs relocate to the endosteum in our model of sublethal radiation-induced thrombocytopenia (4Gy TBI), we asked whether this localization is due to changes in the spatial distribution of the vasculature or to altered microenvironmental SDF-1. In agreement with other TBI models, we find a significant elevation in SDF-1 transcript levels in the marrow at days 1-3 following 4Gy TBI. Radioresistant MKs, which do not decrease in number until after 3 days, have significantly increased CXCR4 surface expression, a finding we also observe following SDF-1 stimulation of MKs both in vitro and in vivo. In situ hybridization was used to localize the spatial distribution of SDF-1 RNA in femoral marrow. At 2 days post-4Gy, a significant SDF-1 gradient develops with 30% higher SDF-1 message adjacent to the endosteum than in the central marrow. However, this gradient is dynamically eliminated 24 hours later at 3 days post-TBI. These shifts in SDF-1 expression are accompanied by parallel changes in the spatial distribution of MKs by immunohistochemistry. At 2 days post-TBI, there is over a 40% increase in MK in the endosteal niche. In contrast, MKs in the endosteal niche decrease by more than 15% at 3 days, coincident with a significant increase in the MKs associated with vascular endothelium. Thus, these data suggest that the spatial distribution of MKs is dependent upon the localization of SDF-1 in the rapidly fluctuating post-injury bone marrow. To determine if SDF-1 functionally contributes to MK niche changes, we stabilized endogenously-produced SDF-1 using Diprotin A, an inhibitor of SDF-1-inactivating protease DPP4. In uninjured marrow, Diprotin A treatment causes over a 30% rise in MK association with vasculature and a 20% increase in circulating platelets 24 hours later, with no change in MK number. Elevation of vascular SDF-1 by intravenous (IV) administration yields similar results. These data indicate that an endogenous SDF-1 gradient toward the vasculature contributes to homeostatic megakaryopoiesis and thrombopoiesis. At 2 days post-TBI, when endosteal SDF-1 message is increased, stabilization with Diprotin A results in a 40% decrease in MKs associated with vasculature and a small but significant decrease in platelets 24 hours later. Further supporting a role for altered SDF-1 gradients, elevating vascular levels with IV SDF-1 at 2 days causes the opposite effect of Diprotin A, with more MKs found in the vascular niche and a rise in peripheral platelet count. In contrast, at 3 days post-TBI, stabilization of endogenous SDF-1 with Diprotin A causes a further 25% increase in MKs in the vascular niche and a 10% rise in circulating platelets, consistent with the rapid loss of the endosteal SDF-1 gradient. Taken together, our data demonstrate that changes in microenvironmental SDF-1 regulate the spatial distribution of MKs in the post-TBI bone marrow. Importantly, the observed SDF-1 changes have functional consequences for platelet production, as the movement of MKs toward the endosteum decreases circulating platelets, while MK association with the vasculature increases circulating platelets. This knowledge will ultimately lead to improved therapeutic strategies to enhance platelet output in the setting of thrombocytopenia and highlights the need to carefully optimize the timing of therapeutic interventions. Disclosures: No relevant conflicts of interest to declare.

Roles of Exosomes in the Hematopoietic Stem Cell-Supporting Capacity of Stromal Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1193-1193
Author(s):  
Gregoire Stik ◽  
Simon Crequit ◽  
Jennifer Durant ◽  
Laurence Petit ◽  
Pierre Charbord ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cross Talk ◽  
Stromal Cells ◽  
Stromal Cell ◽  
High Throughput Sequencing ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Supporting Capacity ◽  
Hematopoietic Stem ◽  
The Cross

Abstract Hematopoietic stem cells (HSCs) are identified by their ability to self-renew and to differentiate into all blood cell lineages. In vivo, hematopoietic stem/progenitor cells (HSPCs) are in close association with stromal cells that constitute a supportive microenvironment also called niche. Recently, exosomes that are small microvesicles enclosed by a lipid bilayer and enriched in cytoplasmic proteins, mRNAs, microRNAs, have emerged as major communication mediators between cells. However, their implication in the cross-talk between HSCs and stromal cells is still largely unknown. This study aims to assess the existence and the functionality of stromal cell-derived exosomes in the HSPC support. To address this issue, we used two murine stromal cell lines derived from the fetal liver and with differing capacity to maintain HSPCs ex vivo as revealed by repopulation assay and long-term cultures. AFT024 (AFT) harbors a potent HSPC supporting capacity in vitro whereas BFC012 (BFC) is non supportive. For each cell line, the exosome fractions were isolated from culture supernatant by ultra-centrifugation. Electron microscopy, western blot, and flow cytometry analyses revealed that both AFT and BFC stromal cells secrete exosomes. Interestingly, using PKH67 stained exosomes, we demonstrated that bone marrow Lin-Sca-1+c-kit+ (LSK) cells preferentially uptake AFT-derived exosomes. This observation might be related to the different tetraspanin compositions of AFT and BFC derived exosomes as observed by flow cytometry. We then showed an increase in cell viability and clonogenic potential when LSK cells were exposed to AFT-derived exosomes for 96 hours in cytokine-free medium as compared to controls. Moreover, cultures with AFT-derived exosomes exhibited a 3.5 fold increase in the number of LSK cells as compared to untreated conditions. We then used high-throughput sequencing to explore the molecular signatures of AFT and BFC derived exosomes, as well as their cells of origin. We identified a list of 394 mRNAs and 6 microRNAs specifically expressed in exosomes and correlated to the HSPC support. Gene ontology analysis revealed that the apoptotic regulation, cell survival and proliferation pathways were significantly enriched in the AFT-derived exosomal signature. In addition, we showed the transfer of mRNAs involved in these pathways from the AFT-exosomes to the LSK recipient cells. Together with our observation of a decrease in the LSK apoptotic cells after co-culture with AFT-derived exosomes, these data suggest that exosomes released by AFT cells may protect HSPCs from apoptosis. Collectively, our results revealed an important role for exosomes in the HSPC supporting capacity of stromal cells. This work provides new insights in our understanding of the molecular and cellular mechanisms involved in the cross-talk between HSPCs and their niches. It may also have interesting applications in regenerative medicine, regarding the ex vivo manipulation of HSCs in stromal-free conditions for cell therapy. Disclosures No relevant conflicts of interest to declare.

scholarly journals Bone Angiogenesis and Vascular Niche Remodeling in Stress, Aging, and Diseases

2020 ◽  
Vol 8 ◽  
Author(s):  
Sina Stucker ◽  
Junyu Chen ◽  
Fiona E. Watt ◽  
Anjali P. Kusumbe
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Cell Types ◽  
Stress Factors ◽  
Hematopoietic Stem ◽  
Perivascular Cell ◽  
Vascular Niche ◽  
Bm Niche ◽  
Highly Sensitive ◽  
Stem And Progenitor Cells

The bone marrow (BM) vascular niche microenvironments harbor stem and progenitor cells of various lineages. Bone angiogenesis is distinct and involves tissue-specific signals. The nurturing vascular niches in the BM are complex and heterogenous consisting of distinct vascular and perivascular cell types that provide crucial signals for the maintenance of stem and progenitor cells. Growing evidence suggests that the BM niche is highly sensitive to stress. Aging, inflammation and other stress factors induce changes in BM niche cells and their crosstalk with tissue cells leading to perturbed hematopoiesis, bone angiogenesis and bone formation. Defining vascular niche remodeling under stress conditions will improve our understanding of the BM vascular niche and its role in homeostasis and disease. Therefore, this review provides an overview of the current understanding of the BM vascular niches for hematopoietic stem cells and their malfunction during aging, bone loss diseases, arthritis and metastasis.

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