ABSTRACT
Platelet dysregulation is drastically increased with advanced age and contributes to making cardiovascular disorders the leading cause of death of elderly humans. Here, we reveal a direct differentiation pathway from hematopoietic stem cells into platelets that is progressively propagated upon aging. Remarkably, the aging-enriched platelet path is decoupled from all other hematopoietic lineages, including erythropoiesis, and operates as an additional layer in parallel with canonical platelet production. This results in two molecularly and functionally distinct populations of megakaryocyte progenitors. The age-induced megakaryocyte progenitors have a profoundly enhanced capacity to engraft, expand, restore, and reconstitute platelets in situ and upon transplantation and produce an additional platelet population in old mice. The two pools of co-existing platelets cause age-related thrombocytosis and dramatically increased thrombosis in vivo. Strikingly, aging-enriched platelets are functionally hyper-reactive compared with the canonical platelet populations. These findings reveal stem cell-based aging as a mechanism for platelet dysregulation and age-induced thrombosis.
Subject(s)
Aging , Blood Platelets , Cell Differentiation , Hematopoietic Stem Cells , Thrombosis , Animals , Hematopoietic Stem Cells/metabolism , Blood Platelets/metabolism , Thrombosis/pathology , Thrombosis/metabolism , Mice , Humans , Megakaryocytes/metabolism , Mice, Inbred C57BL , Megakaryocyte Progenitor Cells/metabolism , MaleABSTRACT
Rare multipotent stem cells replenish millions of blood cells per second through a time-consuming process, passing through multiple stages of increasingly lineage-restricted progenitors. Although insults to the blood-forming system highlight the need for more rapid blood replenishment from stem cells, established models of hematopoiesis implicate only one mandatory differentiation pathway for each blood cell lineage. Here, we establish a nonhierarchical relationship between distinct stem cells that replenish all blood cell lineages and stem cells that replenish almost exclusively platelets, a lineage essential for hemostasis and with important roles in both the innate and adaptive immune systems. These distinct stem cells use cellularly, molecularly and functionally separate pathways for the replenishment of molecularly distinct megakaryocyte-restricted progenitors: a slower steady-state multipotent pathway and a fast-track emergency-activated platelet-restricted pathway. These findings provide a framework for enhancing platelet replenishment in settings in which slow recovery of platelets remains a major clinical challenge.
Subject(s)
Blood Platelets , Cell Differentiation , Hematopoietic Stem Cells , Megakaryocytes , Blood Platelets/immunology , Blood Platelets/metabolism , Animals , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Mice , Cell Differentiation/immunology , Megakaryocytes/cytology , Cell Lineage , Mice, Inbred C57BL , Hematopoiesis , Thrombopoiesis , Mice, Knockout , Humans , Multipotent Stem Cells/cytology , Multipotent Stem Cells/metabolism , Multipotent Stem Cells/immunologyABSTRACT
Emerging evidence has revealed a direct differentiation route from hematopoietic stem cells to megakaryocytes (direct route), in addition to the classical differentiation route through a series of restricted hematopoietic progenitors (stepwise route). This raises the question of the importance of two alternative routes for megakaryopoiesis. Here, we developed fate-mapping systems to distinguish the two routes, comparing their quantitative and functional outputs. We found that megakaryocytes were produced through the two routes with comparable kinetics and quantity under homeostasis. Single-cell RNA sequencing of the fate-mapped megakaryocytes revealed that the direct and stepwise routes contributed to the niche-supporting and immune megakaryocytes, respectively, but contributed to the platelet-producing megakaryocytes together. Megakaryocytes derived from the two routes displayed different activities and were differentially regulated by chemotherapy and inflammation. Our work links differentiation route to the heterogeneity of megakaryocytes. Alternative differentiation routes result in variable combinations of functionally distinct megakaryocyte subpopulations poised for different physiological demands.
Subject(s)
Megakaryocytes , Thrombopoiesis , Cell Differentiation/genetics , Hematopoietic Stem Cells , Blood PlateletsABSTRACT
Platelet homeostasis is essential for vascular integrity and immune defence1,2. Although the process of platelet formation by fragmenting megakaryocytes (MKs; thrombopoiesis) has been extensively studied, the cellular and molecular mechanisms required to constantly replenish the pool of MKs by their progenitor cells (megakaryopoiesis) remains unclear3,4. Here we use intravital imaging to track the cellular dynamics of megakaryopoiesis over days. We identify plasmacytoid dendritic cells (pDCs) as homeostatic sensors that monitor the bone marrow for apoptotic MKs and deliver IFNα to the MK niche triggering local on-demand proliferation and maturation of MK progenitors. This pDC-dependent feedback loop is crucial for MK and platelet homeostasis at steady state and under stress. pDCs are best known for their ability to function as vigilant detectors of viral infection5. We show that virus-induced activation of pDCs interferes with their function as homeostatic sensors of megakaryopoiesis. Consequently, activation of pDCs by SARS-CoV-2 leads to excessive megakaryopoiesis. Together, we identify a pDC-dependent homeostatic circuit that involves innate immune sensing and demand-adapted release of inflammatory mediators to maintain homeostasis of the megakaryocytic lineage.
Subject(s)
Dendritic Cells , Homeostasis , Megakaryocytes , Thrombopoiesis , Animals , Female , Humans , Male , Mice , Apoptosis , Blood Platelets/cytology , Bone Marrow , Cell Lineage , Cell Proliferation , Dendritic Cells/immunology , Dendritic Cells/cytology , Feedback, Physiological , Immunity, Innate , Intravital Microscopy , Megakaryocytes/cytology , Megakaryocytes/immunology , Mice, Inbred C57BL , SARS-CoV-2/immunology , COVID-19/immunology , COVID-19/physiopathology , COVID-19/virologyABSTRACT
In somatic tissue differentiation, chromatin accessibility changes govern priming and precursor commitment towards cellular fates1-3. Therefore, somatic mutations are likely to alter chromatin accessibility patterns, as they disrupt differentiation topologies leading to abnormal clonal outgrowth. However, defining the impact of somatic mutations on the epigenome in human samples is challenging due to admixed mutated and wild-type cells. Here, to chart how somatic mutations disrupt epigenetic landscapes in human clonal outgrowths, we developed genotyping of targeted loci with single-cell chromatin accessibility (GoT-ChA). This high-throughput platform links genotypes to chromatin accessibility at single-cell resolution across thousands of cells within a single assay. We applied GoT-ChA to CD34+ cells from patients with myeloproliferative neoplasms with JAK2V617F-mutated haematopoiesis. Differential accessibility analysis between wild-type and JAK2V617F-mutant progenitors revealed both cell-intrinsic and cell-state-specific shifts within mutant haematopoietic precursors, including cell-intrinsic pro-inflammatory signatures in haematopoietic stem cells, and a distinct profibrotic inflammatory chromatin landscape in megakaryocytic progenitors. Integration of mitochondrial genome profiling and cell-surface protein expression measurement allowed expansion of genotyping onto DOGMA-seq through imputation, enabling single-cell capture of genotypes, chromatin accessibility, RNA expression and cell-surface protein expression. Collectively, we show that the JAK2V617F mutation leads to epigenetic rewiring in a cell-intrinsic and cell type-specific manner, influencing inflammation states and differentiation trajectories. We envision that GoT-ChA will empower broad future investigations of the critical link between somatic mutations and epigenetic alterations across clonal populations in malignant and non-malignant contexts.
Subject(s)
Chromatin , Epigenesis, Genetic , Genotype , Mutation , Single-Cell Analysis , Animals , Female , Humans , Male , Mice , Antigens, CD34/metabolism , Cell Differentiation/genetics , Chromatin/chemistry , Chromatin/genetics , Chromatin/metabolism , Epigenesis, Genetic/genetics , Epigenome/genetics , Genome, Mitochondrial/genetics , Genotyping Techniques , Hematopoiesis/genetics , Hematopoietic Stem Cells/metabolism , Hematopoietic Stem Cells/pathology , Inflammation/genetics , Inflammation/pathology , Janus Kinase 2/genetics , Janus Kinase 2/metabolism , Megakaryocytes/metabolism , Megakaryocytes/pathology , Membrane Proteins/genetics , Myeloproliferative Disorders/genetics , Myeloproliferative Disorders/metabolism , Myeloproliferative Disorders/pathology , RNA/genetics , Clone Cells/metabolismABSTRACT
The bone marrow adjusts blood cell production to meet physiological demands in response to insults. The spatial organization of normal and stress responses are unknown owing to the lack of methods to visualize most steps of blood production. Here we develop strategies to image multipotent haematopoiesis, erythropoiesis and lymphopoiesis in mice. We combine these with imaging of myelopoiesis1 to define the anatomy of normal and stress haematopoiesis. In the steady state, across the skeleton, single stem cells and multipotent progenitors distribute through the marrow enriched near megakaryocytes. Lineage-committed progenitors are recruited to blood vessels, where they contribute to lineage-specific microanatomical structures composed of progenitors and immature cells, which function as the production sites for each major blood lineage. This overall anatomy is resilient to insults, as it was maintained after haemorrhage, systemic bacterial infection and granulocyte colony-stimulating factor (G-CSF) treatment, and during ageing. Production sites enable haematopoietic plasticity as they differentially and selectively modulate their numbers and output in response to insults. We found that stress responses are variable across the skeleton: the tibia and the sternum respond in opposite ways to G-CSF, and the skull does not increase erythropoiesis after haemorrhage. Our studies enable in situ analyses of haematopoiesis, define the anatomy of normal and stress responses, identify discrete microanatomical production sites that confer plasticity to haematopoiesis, and uncover unprecedented heterogeneity of stress responses across the skeleton.
Subject(s)
Hematopoiesis , Hematopoietic Stem Cells , Stress, Physiological , Animals , Female , Male , Mice , Aging/physiology , Bacterial Infections/pathology , Bacterial Infections/physiopathology , Blood Vessels/cytology , Cell Lineage , Erythropoiesis , Granulocyte Colony-Stimulating Factor/metabolism , Hematopoiesis/physiology , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Hemorrhage/pathology , Hemorrhage/physiopathology , Lymphopoiesis , Megakaryocytes/cytology , Multipotent Stem Cells/cytology , Multipotent Stem Cells/metabolism , Myelopoiesis , Skull/blood supply , Skull/pathology , Skull/physiopathology , Sternum/blood supply , Sternum/cytology , Sternum/metabolism , Stress, Physiological/physiology , Tibia/blood supply , Tibia/cytology , Tibia/metabolismABSTRACT
Understanding the regulatory mechanisms facilitating hematopoietic stem cell (HSC) specification during embryogenesis is important for the generation of HSCs in vitro. Megakaryocyte emerged from the yolk sac and produce platelets, which are involved in multiple biological processes, such as preventing hemorrhage. However, whether megakaryocytes regulate HSC development in the embryonic aorta-gonad-mesonephros (AGM) region is unclear. Here, we use platelet factor 4 (PF4)-Cre;Rosa-tdTomato+ cells to report presence of megakaryocytes in the HSC developmental niche. Further, we use the PF4-Cre;Rosa-DTA (DTA) depletion model to reveal that megakaryocytes control HSC specification in the mouse embryos. Megakaryocyte deficiency blocks the generation and maturation of pre-HSCs and alters HSC activity at the AGM. Furthermore, megakaryocytes promote endothelial-to-hematopoietic transition in a OP9-DL1 coculture system. Single-cell RNA-sequencing identifies megakaryocytes positive for the cell surface marker CD226 as the subpopulation with highest potential in promoting the hemogenic fate of endothelial cells by secreting TNFSF14. In line, TNFSF14 treatment rescues hematopoietic cell function in megakaryocyte-depleted cocultures. Taken together, megakaryocytes promote production and maturation of pre-HSCs, acting as a critical microenvironmental control factor during embryonic hematopoiesis.
Subject(s)
Hematopoietic Stem Cells , Megakaryocytes , Animals , Megakaryocytes/cytology , Megakaryocytes/metabolism , Mice , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Cell Differentiation , Hematopoiesis/physiology , Mesonephros/embryology , Mesonephros/metabolism , Mesonephros/cytology , Endothelial Cells/metabolism , Endothelial Cells/cytology , Coculture TechniquesABSTRACT
ABSTRACT: Lysyl oxidase (LOX) is a facilitator of extracellular matrix cross-linking. Using newly developed megakaryocyte-specific LOX knockout mice, we show that LOX expressed in these scarce bone marrow cells affects bone volume and collagen architecture in a sex-dependent manner.
Subject(s)
Megakaryocytes , Mice, Knockout , Protein-Lysine 6-Oxidase , Animals , Protein-Lysine 6-Oxidase/metabolism , Protein-Lysine 6-Oxidase/genetics , Megakaryocytes/metabolism , Megakaryocytes/cytology , Mice , Male , Female , Bone and Bones/metabolism , Sex Characteristics , Collagen/metabolism , Gene Deletion , Sex Factors , Extracellular Matrix ProteinsABSTRACT
Platelets are among the most abundant cells within the circulation. Given that the platelet lifespan is 7 to 10 days in humans, a constant production of around 100 billion platelets per day is required. Platelet production from precursor cells called megakaryocytes is one of the most enigmatic processes in human biology. Although it has been studied for over a century, there is still controversy about the exact mechanisms leading to platelet release into circulation. The formation of proplatelet extensions from megakaryocytes into bone marrow sinusoids is the best-described mechanism explaining the origin of blood platelets. However, using powerful imaging techniques, several emerging studies have recently raised challenging questions in the field, suggesting that small platelet-sized structures called buds might also contribute to the circulating platelet pool. How and whether these structures differ from microvesicles or membrane blebs, which have previously been described to be released from megakaryocytes, is still a matter of discussion. In this review, we will summarize what the past and present have revealed about platelet production and whether mature blood platelets might emerge via different mechanisms.
Subject(s)
Blood Platelets , Megakaryocytes , Thrombopoiesis , Humans , Blood Platelets/metabolism , Megakaryocytes/cytology , Megakaryocytes/metabolism , Animals , Thrombopoiesis/physiologyABSTRACT
PURPOSE OF REVIEW: Single-cell RNA sequencing studies have revealed transcriptional heterogeneity within the megakaryocytic lineage and the identified unique subsets. In this review, we discuss the functional and phenotypic plasticity of these subpopulations as well as the impacts on health and disease. RECENT FINDINGS: Megakaryocytes (MKs) can be transcriptionally categorized into platelet generating, niche supporting, immune, and cycling cells, which are distinguished by their unique gene expression patterns and cellular markers. Additionally, a significant population of these cells has been established to reside in the nonhematopoietic tissues and they display enhanced immune-related characteristics. Combined with the location in which the megakaryocytes exist, these cells can play unique roles dictated by their current environment and biological needs, including responding to changes in pathogen exposure. SUMMARY: Advances in megakaryocyte research has elucidated the existence of multiple subpopulations of MKs that serve different functions. These subpopulations implicate a greater potential for MKs to be regulators of health and suggest new avenues for treatments and therapies in related diseases.
Subject(s)
Blood Platelets , Megakaryocytes , Humans , Megakaryocytes/metabolism , Blood Platelets/metabolismABSTRACT
Megakaryopoiesis and platelet production is a complex process that is underpotential regulation at multiple stages. Many long non-coding RNAs (lncRNAs) are distributed in hematopoietic stem cells and platelets. lncRNAs may play important roles as key epigenetic regulators in megakaryocyte differentiation and proplatelet formation. lncRNA NORAD can affect cell ploidy by sequestering PUMILIO proteins, although its direct effect on megakaryocyte differentiation and thrombopoiesis is still unknown. In this study, we demonstrate NORAD RNA is highly expressed in the cytoplasm during megakaryocyte differentiation. Interestingly, we identified for the first time that NORAD has a strong inhibitory effect on megakaryocyte differentiation and proplatelet formation from cultured megakaryocytes. DUSP6/ERK1/2 pathway is activated in response to NORAD knockdown during megakaryocytopoiesis, which is achieved by sequestering PUM2 proteins. Finally, compared with the wild-type control mice, NORAD knockout mice show a faster platelet recovery after severe thrombocytopenia induced by 6 Gy total body irradiation. These findings demonstrate lncRNA NORAD has a key role in regulating megakaryocyte differentiation and thrombopoiesis, which provides a promising molecular target for the treatment of platelet-related diseases such as severe thrombocytopenia.
Subject(s)
Blood Platelets , Cell Differentiation , Dual Specificity Phosphatase 6 , Megakaryocytes , RNA, Long Noncoding , Thrombopoiesis , Animals , Humans , Mice , Blood Platelets/metabolism , Cell Differentiation/genetics , Cells, Cultured , Dual Specificity Phosphatase 6/metabolism , Dual Specificity Phosphatase 6/genetics , MAP Kinase Signaling System , Megakaryocytes/metabolism , Megakaryocytes/cytology , Mice, Inbred C57BL , Mice, Knockout , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , Thrombocytopenia/genetics , Thrombocytopenia/metabolism , Thrombocytopenia/pathology , Thrombopoiesis/geneticsABSTRACT
Thrombocytopenia is a critical complication after radiation therapy and exposure. Dysfunction of megakaryocyte development and platelet production are key pathophysiological stages in ionizing radiation (IR)-induced thrombocytopenia. Protein kinase C (PKC) plays an important role in regulating megakaryocyte development and platelet production. However, it remains unclear how PKC regulates IR-induced megakaryocyte apoptosis. In this study, we found that pretreatment of PKC pan-inhibitor Go6983 delayed IR-induced megakaryocyte apoptosis, and inhibited IR-induced mitochondrial membrane potential and ROS production in CMK cells. Moreover, suppressing PKC activation inhibited cleaved caspase3 expression and reduced p38 phosphorylation levels, and IR-induced PKC activation might be regulated by p53. In vivo experiments confirmed that Go6983 promoted platelet count recovery after 21 days of 3 Gy total body irradiation. Furthermore, Go6983 reduced megakaryocyte apoptosis, increased the number of megakaryocyte and polyploid formation in bone marrow, and improved the survival rate of 6 Gy total body irradiation. In conclusion, our results provided a potential therapeutic target for IR-induced thrombocytopenia.
Subject(s)
Megakaryocytes , Thrombocytopenia , Humans , Protein Kinase C/metabolism , Protein Kinase C/therapeutic use , X-Rays , Thrombocytopenia/etiology , Thrombopoiesis , Apoptosis , Blood PlateletsABSTRACT
TAFRO syndrome is a rare systemic inflammatory disorder of unknown etiology characterized by thrombocytopenia, anasarca, fever, reticulin myelofibrosis, renal dysfunction, and organomegaly. The diagnosis of TAFRO syndrome can be challenging; however, prompt diagnosis is vital because TAFRO syndrome is a progressive and life-threatening disease. We have showcased five patients with TAFRO syndrome who had similar bone marrow (BM) findings that could be considered the findings that characterize TAFRO syndrome. All patients were treated with corticosteroids and tocilizumab; three of the five patients (60 %) responded positively to the treatment. The unique BM findings observed in this study were megakaryocytes with distinct multinuclei and three-dimensional and indistinct bizarre nuclei ("dysmorphic megakaryocyte"), similar to the megakaryocyte morphology observed in myeloproliferative neoplasms (MPNs). Notably, dysmorphic megakaryocytes were observed in all five cases, whereas only two of the five patients tested positive for reticulin myelofibrosis, and three of the five patients had megakaryocytic hyperplasia, which are considered typical findings of TAFRO syndrome. Thus, the BM findings of dysmorphic megakaryocytes could help in the correct and immediate diagnosis of TAFRO syndrome.
Subject(s)
Megakaryocytes , Humans , Megakaryocytes/pathology , Male , Female , Middle Aged , Adult , Bone Marrow/pathology , Castleman Disease/pathology , Castleman Disease/diagnosis , Aged , Thrombocytopenia/pathology , Thrombocytopenia/diagnosis , Primary Myelofibrosis/pathology , Primary Myelofibrosis/diagnosisABSTRACT
BACKGROUND: Circular RNAs (circRNA) are pivotal in hematological diseases. Previous study showed that circ_0014614 (circDAP3) was significantly underexpressed in bone marrow-derived exosomes from essential thrombocythemia (ET) patients, affecting the differentiation of bone marrow lineage cells into megakaryocytes. METHODS: Fluorescence in situ hybridization (FISH) was used to display circ_0014614's primary cytoplasmic location in K562 cells. Cytoscape software was used to predict the circRNA-miRNA-mRNA networks, and their expression at the cellular level was detected by Quantitative reverse transcription-polymerase chain reaction (qRT-PCR). qRT-PCR was utilized to detect the expression levels of circ_0014614ï¼miR-138-5p and caspase3 mRNA. Western blot was used to determine the protein levels of GATA-1, RUNX-1, NF-E2, CD41 and caspase3. The proliferation of K562 cells was assessed using the Cell Counting Kit-8 (CCK-8) Assay. Furthermore, the interplay between miR-138-5p and circ_0014614 or caspase3 was elucidated through a Dual-luciferase reporter assay. RESULTS: FISH assay indicated circ_0014614's primary cytoplasmic location in K562 cells. In ET bone marrow and K562 cells, circ_0014614 and caspase3 were down-regulated, whereas miR-138-5p saw a significant surge. Overexpressing circ_0014614 curtailed K562 cells' proliferation and differentiation. Further, circ_0014614 targeted miR-138-5p, with heightened miR-138-5p levels counteracting circ_0014614's inhibition. MiR-138-5p further targeted caspase3, and caspase3 silencing neutralized suppressed miR-138-5p's effects on K562 cell differentiation. CONCLUSION: Circ_0014614 was down-regulated in ET bone marrow and bone marrow lineage cells, and upregulating circ_0014614 can inhibit bone marrow lineage cells' proliferation and differentiation into megakaryocytes. Mechanistically, circ_0014614 functioned as ceRNA via sponging miR-138-5p and alleviated the inhibitory effect of miR-138-5p on its target caspase3, which potentially deters tumor activity in ET.
Subject(s)
Caspase 3 , Cell Differentiation , Megakaryocytes , MicroRNAs , RNA, Circular , Thrombocythemia, Essential , Humans , MicroRNAs/genetics , MicroRNAs/metabolism , Megakaryocytes/metabolism , Megakaryocytes/pathology , RNA, Circular/genetics , Caspase 3/metabolism , Thrombocythemia, Essential/genetics , Thrombocythemia, Essential/pathology , Thrombocythemia, Essential/metabolism , K562 Cells , Bone Marrow Cells/metabolism , Bone Marrow Cells/pathology , Female , Male , Middle AgedABSTRACT
Thrombopoiesis is the production of platelets from megakaryocytes in the bone marrow of mammals. In fish, thrombopoiesis involves the formation of thrombocytes without megakaryocyte-like precursors but derived from erythrocyte thrombocyte bi-functional precursor cells. One unique feature of thrombocyte differentiation involves the maturation of young thrombocytes in circulation. In this study, we investigated the role of hox genes in zebrafish thrombopoiesis to model platelet production. We selected hoxa10b, hoxb2a, hoxc5a, hoxd3a, and hoxc11b from thrombocyte RNA expression data, and checked whether they are expressed in young or mature thrombocytes. We found hoxa10b, hoxb2a, hoxc5a, and hoxd3a were expressed in both young and mature thrombocytes and hoxc11b was expressed in only young thrombocytes. We then performed knockdowns of these 5 hox genes and found hoxc11b knockdown resulted in thrombocytosis and the rest showed thrombocytopenia. To identify hox genes that could have been missed by the above datasets, we performed knockdowns 47 hox genes in the zebrafish genome and found hoxa9a, and hoxb1a knockdowns resulted in thrombocytopenia and they were expressed in both young and mature thrombocytes. In conclusion, our comprehensive knockdown study identified Hoxa10b, Hoxb2a, Hoxc5a, Hoxd3a, Hoxa9a, and Hoxb1a, as positive regulators and Hoxc11b, as a negative regulator for thrombocyte development.
Subject(s)
Thrombocytopenia , Thrombopoiesis , Animals , Thrombopoiesis/genetics , Zebrafish/genetics , Zebrafish/metabolism , Genes, Homeobox , Blood Platelets/metabolism , Megakaryocytes , Thrombocytopenia/genetics , Mammals/geneticsABSTRACT
Immune thrombocytopenia (ITP) is an autoimmune disease caused by the loss of immune tolerance to platelet autoantigens, resulting in reduced platelet production and increased platelet destruction. Impaired megakaryocyte differentiation and maturation is a key factor in the pathogenesis and treatment of ITP. Sarcandra glabra, a plant of the Chloranthaceae family, is commonly used in clinical practice to treat ITP, and daucosterol (Dau) is one of its active ingredients. However, whether Dau can treat ITP and the key mechanism of its effect are still unclear. In this study, we found that Dau could effectively promote the differentiation and maturation of megakaryocytes and the formation of polyploidy in the megakaryocyte differentiation disorder model constructed by co-culturing Dami and HS-5 cells. In vivo experiments showed that Dau could not only increase the number of polyploidized megakaryocytes in the ITP rat model, but also promote the recovery of platelet count. In addition, through network pharmacology analysis, we speculated that the JAK2-STAT3 signaling pathway might be involved in the process of Dau promoting megakaryocyte differentiation. Western blot results showed that Dau inhibited the expression of P-JAK2 and P-STAT3. In summary, these results provide a basis for further studying the pharmacological mechanism of Dau in treating ITP.
Subject(s)
Cell Differentiation , Janus Kinase 2 , Megakaryocytes , Purpura, Thrombocytopenic, Idiopathic , STAT3 Transcription Factor , Signal Transduction , Animals , Humans , Male , Rats , Cell Differentiation/drug effects , Disease Models, Animal , Janus Kinase 2/metabolism , Megakaryocytes/metabolism , Megakaryocytes/drug effects , Megakaryocytes/cytology , Purpura, Thrombocytopenic, Idiopathic/metabolism , Purpura, Thrombocytopenic, Idiopathic/drug therapy , Purpura, Thrombocytopenic, Idiopathic/pathology , Signal Transduction/drug effects , Sitosterols/pharmacology , STAT3 Transcription Factor/metabolismABSTRACT
Epidemiological studies show that cardiovascular events related to platelet hyperactivity remain the leading causes of death among multiple sclerosis (MS) patients. Quantitative or structural changes of platelet cytoskeleton alter their morphology and function. Here, we demonstrated, for the first time, the structural changes in MS platelets that may be related to their hyperactivity. MS platelets were found to form large aggregates compared to control platelets. In contrast to the control, the images of overactivated, irregularly shaped MS platelets show changes in the cytoskeleton architecture, fragmented microtubule rings. Furthermore, MS platelets have long and numerous pseudopodia rich in actin filaments. We showed that MS platelets and megakaryocytes, overexpress ß1-tubulin and ß-actin mRNAs and proteins and have altered post-translational modification patterns. Moreover, we identified two previously undisclosed mutations in the gene encoding ß1-tubulin in MS. We propose that the demonstrated structural changes of platelet cytoskeleton enhance their ability to adhere, aggregate, and degranulate fueling the risk of adverse cardiovascular events in MS.
Subject(s)
Blood Platelets , Cytoskeletal Proteins , Cytoskeleton , Multiple Sclerosis , Tubulin , Humans , Multiple Sclerosis/metabolism , Multiple Sclerosis/pathology , Multiple Sclerosis/blood , Blood Platelets/metabolism , Tubulin/metabolism , Tubulin/genetics , Female , Cytoskeleton/metabolism , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , Adult , Male , Middle Aged , Actins/metabolism , Actins/genetics , Megakaryocytes/metabolism , Megakaryocytes/pathology , Protein Processing, Post-Translational , MutationABSTRACT
Variants in the 5' UTR of ANKRD26 are a common cause of inherited thrombocytopenia (ANKRD26-RT), and are associated with sustained ANKRD26 expression, which inhibits megakaryocyte maturation and proplatelet formation. ANKRD26 expression is controlled by the binding of a RUNX1/FLI1 complex to the 5' UTR. To date, all reported ANKRD26-RD associated variants have been within the RUNX1 binding site and a 22 base pair flanking region. Here, we report a novel variant in the 5' UTR of ANKRD26, c.-107C>T. This variant is in the FLI1 binding site, and is predicted to disrupt FLI1 binding due to loss of a hydrogen bond with FLI1. Differentiated PBMCs from affected family members showed impaired megakaryocyte maturation and proplatelet formation and sustained expression of ANKRD26, and platelets from affected family members had higher ANKRD26 expression than control platelets. The variant increased activity of the ANKRD26 promotor in a reporter assay. We also provide evidence that the previously reported c.-140C>G ANKRD26 5' UTR variant is benign and not associated with thrombocytopenia. Identification of the c.-107C>T variant extends the range of the regulatory region in the 5' UTR of ANKRD26 that is associated with ANKRD26-RT.
Subject(s)
5' Untranslated Regions , Proto-Oncogene Protein c-fli-1 , Thrombocytopenia , Humans , Thrombocytopenia/genetics , Thrombocytopenia/pathology , 5' Untranslated Regions/genetics , Binding Sites , Proto-Oncogene Protein c-fli-1/genetics , Proto-Oncogene Protein c-fli-1/metabolism , Male , Female , Pedigree , Megakaryocytes/metabolism , Megakaryocytes/pathology , Blood Platelets/metabolism , Blood Platelets/pathology , Core Binding Factor Alpha 2 Subunit/genetics , Protein Binding , Genetic Predisposition to Disease , Intercellular Signaling Peptides and ProteinsABSTRACT
BACKGROUND: Hematopoietic stem cell (HSC) regeneration underlies hematopoietic recovery from myelosuppression, which is a life-threatening side effect of cytotoxicity. HSC niche is profoundly disrupted after myelosuppressive injury, while if and how the niche is reshaped and regulates HSC regeneration are poorly understood. METHODS: A mouse model of radiation injury-induced myelosuppression was built by exposing mice to a sublethal dose of ionizing radiation. The dynamic changes in the number, distribution and functionality of HSCs and megakaryocytes were determined by flow cytometry, immunofluorescence, colony assay and bone marrow transplantation, in combination with transcriptomic analysis. The communication between HSCs and megakaryocytes was determined using a coculture system and adoptive transfer. The signaling mechanism was investigated both in vivo and in vitro, and was consolidated using megakaryocyte-specific knockout mice and transgenic mice. RESULTS: Megakaryocytes become a predominant component of HSC niche and localize closer to HSCs after radiation injury. Meanwhile, transient insulin-like growth factor 1 (IGF1) hypersecretion is predominantly provoked in megakaryocytes after radiation injury, whereas HSCs regenerate paralleling megakaryocytic IGF1 hypersecretion. Mechanistically, HSCs are particularly susceptible to megakaryocytic IGF1 hypersecretion, and mTOR downstream of IGF1 signaling not only promotes activation including proliferation and mitochondrial oxidative metabolism of HSCs, but also inhibits ferritinophagy to restrict HSC ferroptosis. Consequently, the delicate coordination between proliferation, mitochondrial oxidative metabolism and ferroptosis ensures functional HSC expansion after radiation injury. Importantly, punctual IGF1 administration simultaneously promotes HSC regeneration and hematopoietic recovery after radiation injury, representing a superior therapeutic approach for myelosuppression. CONCLUSIONS: Our study identifies megakaryocytes as a last line of defense against myelosuppressive injury and megakaryocytic IGF1 as a novel niche signal safeguarding HSC regeneration.
Subject(s)
Ferroptosis , Hematopoietic Stem Cells , Insulin-Like Growth Factor I , Megakaryocytes , Regeneration , Animals , Hematopoietic Stem Cells/metabolism , Megakaryocytes/metabolism , Megakaryocytes/radiation effects , Insulin-Like Growth Factor I/metabolism , Insulin-Like Growth Factor I/genetics , Ferroptosis/genetics , Mice , Mice, Inbred C57BL , Radiation Injuries/metabolism , Radiation Injuries/pathology , Radiation Injuries/genetics , Signal Transduction/radiation effectsABSTRACT
Mechanisms through which mature megakaryocytes (Mks) and their progenitors sense the bone marrow extracellular matrix to promote lineage differentiation in health and disease are still partially understood. We found PIEZO1, a mechanosensitive cation channel, to be expressed in mouse and human Mks. Human mutations in PIEZO1 have been described to be associated with blood cell disorders. Yet, a role for PIEZO1 in megakaryopoiesis and proplatelet formation has never been investigated. Here, we show that activation of PIEZO1 increases the number of immature Mks in mice, while the number of mature Mks and Mk ploidy level are reduced. Piezo1/2 knockout mice show an increase in Mk size and platelet count, both at basal state and upon marrow regeneration. Similarly, in human samples, PIEZO1 is expressed during megakaryopoiesis. Its activation reduces Mk size, ploidy, maturation, and proplatelet extension. Resulting effects of PIEZO1 activation on Mks resemble the profile in Primary Myelofibrosis (PMF). Intriguingly, Mks derived from Jak2V617F PMF mice show significantly elevated PIEZO1 expression, compared to wild-type controls. Accordingly, Mks isolated from bone marrow aspirates of JAK2V617F PMF patients show increased PIEZO1 expression compared to Essential Thrombocythemia. Most importantly, PIEZO1 expression in bone marrow Mks is inversely correlated with patient platelet count. The ploidy, maturation, and proplatelet formation of Mks from JAK2V617F PMF patients are rescued upon PIEZO1 inhibition. Together, our data suggest that PIEZO1 places a brake on Mk maturation and platelet formation in physiology, and its upregulation in PMF Mks might contribute to aggravating some hallmarks of the disease.