Mouse Bone Marrow Megakaryocytes Research Articles

Spatiotemporal Impact of Sars-Cov-2 Infection on the Transcriptome of Bone Marrow Megakaryocytes

Megakaryocytes, integral to platelet production, predominantly reside in the bone marrow, undergoing regulated fragmentation within sinusoid vessels to release platelets into the bloodstream. Inflammatory states and infections have been shown to influence megakaryocyte transcription, potentially affecting platelet functionality. Notably, COVID-19 has been associated with altered platelet transcriptome. In this study, we hypothesized that SARS-CoV-2 infection could impact the transcriptome of bone marrow megakaryocytes, particularly those in direct proximity to the blood. Utilizing spatial transcriptomic analysis and machine learning techniques, we observed that the transcriptome of healthy mouse bone marrow megakaryocytes exhibited minimal alteration based on proximity to sinusoid vessels. Similar findings were observed during peak SARS-CoV-2 viremia, when the disease primarily affected the lungs. Conversely, a significant divergence in the transcriptome of megakaryocytes was observed during systemic inflammation, even when SARS-CoV-2 was no longer detectable in the lungs or bone marrow. Under these conditions, the transcriptional landscape was enriched in genes associated with structural and translational activities, platelet degranulation, netosis, and auto-immunity. Of the approximately ~19,000 genes identified in megakaryocytes during systemic inflammation, machine learning pinpointed 30 genes significantly altered in cells closely adjacent to sinusoid vessels, with gene ontology attributing these changes primarily to protein deSUMOylation. Intriguingly, the type-I interferon signature and calprotectin (S100A8/A9) were not induced under any condition. Inflammatory cytokines were elevated in the blood of COVID-19 mice, but not in bone marrow plasma, suggesting a preferential impact of inflammation on this specific subset of cells. Collectively, our data indicate that distinct subpopulations of bone marrow megakaryocytes may emerge based on spatial localization and the stage of COVID-19 pathogenesis.

Megakaryocyte polyploidy is inhibited by lysyl oxidase propeptide

Megakaryocytes (MKs), the platelet precursors, undergo an endomitotic cell cycle that leads to polyploidy. Lysyl oxidase propeptide (LOX-PP) is generated from lysyl oxidase (LOX) pro-enzyme after proteolytical cleavage. We recently reported that LOX, a known matrix cross-linking enzyme, contributes to MK lineage expansion. In addition, LOX expression levels are ploidy-dependent, with polyploidy MKs having minimal levels. This led us to test the effects of LOX-PP on the number and ploidy of primary MKs. LOX-PP significantly decreases mouse bone marrow MK ploidy coupled with a reduction in MK size. MK number is unchanged upon LOX-PP treatment. Analysis of LOX-PP- or vehicle-treated MKs by western blotting revealed a reduction in ERK1/2 phosphorylation and in the levels of its downstream targets, cyclin D3 and cyclin E, which are known to play a central role in MK endomitosis. Pull-down assays and immunochemistry staining indicated that LOX-PP interacts with α-tubulin and the mictotubules, which can contribute to decreased MK ploidy. Thus, our findings defined a role for LOX-PP in reducing MK ploidy. This suggests that high-level expression of LOX in aberrantly proliferating MKs could play a part in inhibiting their polyploidization via LOX-PP.

Aberrant quantity and localization of Aurora-B/AIM-1 and survivin during megakaryocyte polyploidization and the consequences of Aurora-B/AIM-1–deregulated expression

AbstractMegakaryocytes skip late anaphase and cytokinesis during endomitosis. We found normal expression and localization of a fundamental regulator of mitosis, Aurora-B/AIM-1, during prophase in polyploidizing mouse bone marrow megakaryocytes. At late anaphase, however, Aurora-B/AIM-1 is absent or mislocalized. Megakaryocytes treated with a proteasome inhibitor display Aurora-B/AIM-1 properly expressed and localized to the midzone, suggesting that protein degradation contributes to this atypical appearance. In contrast, survivin, an Aurora-B/AIM-1 coregulator of mitosis, is not detected at any stage of the endomitotic cell cycle, and in most megakaryocytes proteasome inhibition does not rescue this phenotype. To further explore the importance of reduced Aurora-B/AIM-1 for polyploidization, it was overexpressed in megakaryocytes of transgenic mice. The phenotype includes increased transgenic mRNA, but not protein, in polyploidy megakaryocytes, further suggesting that Aurora-B/AIM-1 is regulated at the protein level. Aurora-B/AIM-1 protein is, however, elevated in diploid transgenic megakaryocytes. Transgenic mice also exhibit enhanced numbers of megakaryocytes with increased proliferative potential, and some mice exhibit mild decreases in ploidy level. Hence, the molecular programming involved in endomitosis is characterized by the mislocalization or absence of at least 2 critical mitotic regulators, Aurora-B/AIM-1 and survivin. Future studies will examine the impact of survivin restoration on mouse megakaryocyte polyploidization.

Thrombopoietin‐induced CXC chemokines, NAP‐2 and PF4, suppress polyploidization and proplatelet formation during megakaryocyte maturation

We previously reported that the expressions of two CXC chemokines, neutrophil activating peptide-2 (NAP-2) and platelet factor-4 (PF-4), were induced by megakaryocyte-specific cytokine thrombopoietin (TPO) in mouse bone marrow megakaryocytes. The roles of these chemokines on megakaryocyte maturation/differentiation processes, including polyploidization and proplatelet formation (PPF) remain unresolved. NAP-2 and PF-4 suppressed the PPF of mature megakaryocytes freshly prepared from mouse bone marrow as well as that of the megakaryocyte progenitors, c-Kit+CD41+ cells, isolated from mouse bone marrow and cultured with TPO. NAP-2 and PF-4 inhibited polyploidization of c-Kit+CD41+ cells in the presence of TPO, and also inhibited the proliferation of c-Kit+CD41+ cells. NAP-2 and PF-4 produced by TPO stimulation in megakaryocytes suppress megakaryocyte maturation and proliferation as a feedback control.

Isolation and Characterization of Mouse Homolog of the Neutrophil Activating Peptide-2

In the presence of thrombopoietin (TPO), megakaryocytes mature by polyploidization and cytoplasmic maturation, and the matured megakaryocytes induce drastic morphological change and proplatelet formation and release a number of platelets. However, the regulatory mechanism of this unique differentiation process is still obscure. We therefore attempted to identify the factors, expression of which is induced by TPO stimulation in mouse bone marrow megakaryocytes. We isolated the mouse homolog of the neutrophil activating peptide-2 (NAP-2). Mouse NAP-2 cDNA encodes a predicted sequence of 113 amino acids and contains the Cys motif (CXC) found in other members of the alpha-chemokine family. At the amino acid level, the predicted mouse NAP-2 has 50.4%, 51.8%, and 72.6% identity with the predicted human, pig, and rat NAP-2, respectively. Northern blot analysis demonstrates that mouse NAP-2 is expressed only in spleen. Furthermore, the RT-PCR technique shows that the mouse NAP-2 gene is clearly upregulated by TPO stimulation in mouse megakaryocytes.

Thrombopoietin-induced polyploidization of bone marrow megakaryocytes is due to a unique regulatory mechanism in late mitosis.

Megakaryocytes undergo a unique differentiation program, becoming polyploid through repeated cycles of DNA synthesis without concomitant cell division. However, the mechanism underlying this polyploidization remains totally unknown. It has been postulated that polyploidization is due to a skipping of mitosis after each round of DNA replication. We carried out immunohistochemical studies on mouse bone marrow megakaryocytes during thrombopoietin- induced polyploidization and found that during this process megakaryocytes indeed enter mitosis and progress through normal prophase, prometaphase, metaphase, and up to anaphase A, but not to anaphase B, telophase, or cytokinesis. It was clearly observed that multiple spindle poles were formed as the polyploid megakaryocytes entered mitosis; the nuclear membrane broke down during prophase; the sister chromatids were aligned on a multifaced plate, and the centrosomes were symmetrically located on either side of each face of the plate at metaphase; and a set of sister chromatids moved into the multiple centrosomes during anaphase A. We further noted that the pair of spindle poles in anaphase were located in close proximity to each other, probably because of the lack of outward movement of spindle poles during anaphase B. Thus, the reassembling nuclear envelope may enclose all the sister chromatids in a single nucleus at anaphase and then skip telophase and cytokinesis. These observations clearly indicate that polyploidization of megakaryocytes is not simply due to a skipping of mitosis, and that the megakaryocytes must have a unique regulatory mechanism in anaphase, e.g., factors regulating anaphase such as microtubule motor proteins might be involved in this polyploidization process.

Overexpression of granulocyte colony-stimulating factor in vivo decreases the level of polyploidization of mouse bone marrow megakaryocytes.

The in vivo effect of G-CSF on the maturation of mouse bone marrow megakaryocytes was studied by monitoring the DNA contents. Megakaryocytes were first identified by a specific 1C2 monoclonal antibody against mouse platelets and megakaryocytes and DNA contents of these cells were measured by propidium iodine. Megakaryocytes of mice transgenic for human G-CSF had a modal DNA class of 8N, showing a striking contrast to the previous reports that normal mouse megakaryocytes from most strains have 16N DNA content as a modal class. Daily 10 micrograms administration of G-CSF to mice for three to five days affected the DNA distribution pattern of bone marrow megakaryocytes, with a higher proportion of cells having 8N DNA contents. This G-CSF treatment, however, did not influence the peripheral blood platelet count or bone marrow megakaryocyte number. Administration of G-CSF along with thrombopoietin (TPO) reduced the proportion of megakaryocytes, with 32N DNA, the DNA class that was increased by TPO. Finally, the presence of mRNA for the mouse G-CSF receptor was demonstrated in two megakaryoblastic cell lines by reverse transcriptase polymerase chain reaction. These results indicated that G-CSF may have a suppressive effect on the maturation of mouse bone marrow megakaryocytes when monitored by the DNA polyploidy. Although further study is clearly necessary, the presence of mRNA for the G-CSF receptor in megakaryocytic lineage strongly suggests the direct action of G-CSF on this cell lineage.

Cyclic nucleotide-dependent regulation of agonist-induced calcium increases in mouse megakaryocytes.

1. The regulatory effects of cyclic AMP and cyclic GMP on ADP- and thrombin-induced increases in [Ca2+]i were studied in mouse bone marrow megakaryocytes. Changes in [Ca2+]i were continuously monitored in single Fura-2-loaded cells using microspectrofluorometry, and cyclic nucleotides were directly introduced into the single cells using the whole-cell patch-clamp technique. 2. ADP increased [Ca2+]i in a concentration-dependent fashion, and its threshold concentration was in the order of 0.01 microM. A low dose of ADP (below 0.1 microM) induced a transient response of [Ca2+]i which recovered to original levels during the stimulation. A high dose of ADP (0.3-10 microM) induced a biphasic response of [Ca2+]i with an initial peak and a plateau lasting until the end of the stimulation. Repeated stimulation with the same dose of ADP induced a reduced response, probably as a result of desensitization. 3. Thrombin increased [Ca2+]i in a concentration-dependent manner. The time courses of the responses were different from those caused by ADP. Thrombin-induced responses lacked the initial sharp peak observed in ADP-induced responses, and caused a sustained response. 4. The ADP-induced increase in [Ca2+]i was antagonized by the presence of prostaglandin E1 (PGE1, 100-1000 nM), in the medium, and by direct injection of cyclic AMP (100-500 microM) or cyclic GMP (500 microM) into the megakaryocyte. When 500 microM-cyclic AMP was injected into the cells, the rise of [Ca2+]i induced by ADP was reduced by 85%. Effects of these antagonists were inhibited by treatment with a protein kinase inhibitor, H-8. Thrombin-induced increases in [Ca2+]i were reduced by direct injection of cyclic AMP or cyclic GMP. 5. ADP could induce an increase in [Ca2+]i in the absence of external Ca2+. The time course of the response was essentially similar to that observed in the normal condition (1 mM-CaCl2), but the size of the response was reduced by 33%. Thus, 67% of the rise in [Ca2+]i induced by ADP could be accounted for by calcium mobilization from internal storage pools. The presence of NiCl2 (5 mM) duplicated the effects of external Ca2+ removal, suggesting the involvement of a Ca2+ influx pathway, which could be inhibited by Ni2+ in ADP stimulation. 6. Injection of cyclic AMP or cyclic GMP reduced ADP-induced increases in [Ca2+]i under conditions of inhibited Ca2+ influx by NiCl2 (5 mM).(ABSTRACT TRUNCATED AT 400 WORDS)

Localization of high concentrations of phosphotyrosine-modified proteins in mouse megakaryocytes

Phosphorylation of tyrosine residues of cellular proteins is a rare event and is considered to be related to the regulation of cellular growth, differentiation, and some forms of neoplastic transformation. Using high-affinity antibodies specific to phosphotyrosine (P-Tyr), we have shown the presence at high concentrations of P-Tyr-modified proteins in mouse bone-marrow megakaryocytes. Immunofluorescence microscopy of semithin frozen sections revealed that P-Tyr labeling was localized in a punctate pattern in the majority of the cytoplasm. The thin outer rim of the cytoplasm and the cell membrane was devoid of the label. Immunogold electron microscopy of ultrathin frozen sections showed that P-Tyr labeling was concentrated mostly on the membranes of the vesicles in the cytoplasm. The membrane demarcation system characteristic of megakaryocytes was not labeled. The intensity of P-Tyr labeling varied from one megakaryocyte to another. These results suggest that tyrosine phosphorylation of specific proteins might be correlated with the developmental stage of megakaryocytes, possibly related to the formation and deposition of the granules.

Localization of High Concentrations of Phosphotyrosine-Modified Proteins in Mouse Megakaryocytes

Abstract Phosphorylation of tyrosine residues of cellular proteins is a rare event and is considered to be related to the regulation of cellular growth, differentiation, and some forms of neoplastic transformation. Using high-affinity antibodies specific to phosphotyrosine (P-Tyr), we have shown the presence at high concentrations of P-Tyr-modified proteins in mouse bone-marrow megakaryocytes. Immunofluorescence microscopy of semithin frozen sections revealed that P-Tyr labeling was localized in a punctate pattern in the majority of the cytoplasm. The thin outer rim of the cytoplasm and the cell membrane was devoid of the label. Immunogold electron microscopy of ultrathin frozen sections showed that P-Tyr labeling was concentrated mostly on the membranes of the vesicles in the cytoplasm. The membrane demarcation system characteristic of megakaryocytes was not labeled. The intensity of P- Tyr labeling varied from one megakaryocyte to another. These results suggest that tyrosine phosphorylation of specific proteins might be correlated with the developmental stage of megakaryocytes, possibly related to the formation and deposition of the granules.

Immunohistochemical localization of membrane and alpha-granule proteins in plastic-embedded mouse bone marrow megakaryocytes and murine megakaryocyte colonies

Using an immunoperoxidase technique that permits optimal antigen localization at the light microscope level, we have detected two platelet alpha-granule constituents and three platelet membrane glycoproteins in mouse bone marrow megakaryocytes and in murine megakaryocyte colonies grown in soft agar culture for three to seven days. Using polyclonal antibodies prepared against human platelet proteins, we have demonstrated labeling for von Willebrand factor, fibrinogen, and the membrane glycoproteins IIIa and GMP-140 in both bone marrow megakaryocytes and megakaryocyte colonies after seven days of culture. Using monoclonal antibodies to membrane glycoproteins IIb and GMP-140, we have demonstrated label in mouse bone marrow megakaryocytes. Granulocyte and macrophage colonies were negative for each of these markers. Murine bone marrow megakaryocytes and megakaryocyte colonies demonstrated a similar enzyme histochemical pattern: weakly positive for alpha-naphthyl acetate esterase and negative for chloroacetate esterase. These data indicate that megakaryocytes grown in soft agar culture express many of the same glycoproteins as bone marrow megakaryocytes. Furthermore, the ability of antibodies directed against human platelet membrane glycoproteins to identify murine megakaryocyte glycoproteins indicates that these constituents have been highly conserved during evolution.

Immunohistochemical Localization of Membrane and a-Granule Proteins in Plastic-Embedded Mouse Bone Marrow Megakaryocytes and Murine Megakaryocyte Colonies

Abstract Using an immunoperoxidase technique that permits optimal antigen localization at the light microscope level, we have detected two platelet alpha-granule constituents and three platelet membrane glycoproteins in mouse bone marrow megakaryocytes and in murine megakaryocyte colonies grown in soft agar culture for three to seven days. Using polyclonal antibodies prepared against human platelet proteins, we have demonstrated labeling for von Willebrand factor, fibrinogen, and the membrane glycoproteins IIIa and GMP-140 in both bone marrow megakaryocytes and megakaryocyte colonies after seven days of culture. Using monoclonal antibodies to membrane glycoproteins IIb and GMP-140, we have demonstrated label in mouse bone marrow megakaryocytes. Granulocyte and macrophage colonies were negative for each of these markers. Murine bone marrow megakaryocytes and megakaryocyte colonies demonstrated a similar enzyme histochemical pattern: weakly positive for alpha-naphthyl acetate esterase and negative for chloroacetate esterase. These data indicate that megakaryocytes grown in soft agar culture express many of the same glycoproteins as bone marrow megakaryocytes. Furthermore, the ability of antibodies directed against human platelet membrane glycoproteins to identify murine megakaryocyte glycoproteins indicates that these constituents have been highly conserved during evolution.

Separation of megakaryocytes from mouse bone marrow by density gradient centrifugation

The density profile of mouse bone marrow megakaryocytes, as determined by discontinuous albumin density gradient centrifugation, was characterized by a single density population (1.088 to 1.119 g/ml) with a peak density of 1.10 g/ml and a maximum enrichment of 15.5. This single population comprised both mature and immature megakaryocytes. The density profile of small, acetylcholinesterase-positive cells, a class of potential megakaryocyte precursors, was almost identical to that of morphologically recognizable megakaryocytes.

Separation of Megakaryocytes From Mouse Bone Marrow by Density Gradient Centrifugation

The density profile of mouse bone marrow megakaryocytes, as determined by discontinuous albumin density gradient centrifugation, was characterized by a single density population (1.088 to 1.119 g/ml) with a peak density of 1.10 g/ml and a maximum enrichment of 15.5. This single population comprised both mature and immature megakaryocytes. The density profile of small, acetylcholinesterase-positive cells, a class of potential megakaryocyte precursors, was almost identical to that of morphologically recognizable megakaryocytes.

Separation of Megakaryocytes From Mouse Bone Marrow by Velocity Sedimentation

Abstract A technique for separating mouse bone marrow megakaryocytes by unit gravity velocity sedimentation is described using a modified sedimentation chamber. The velocity sedimentation profile for megakaryocytes comprises three peaks corresponding to sedimentation velocities of 30, 60, and 100 mm/hr. Morphologic examination of megakaryocytes in the 30 mm/hr peak reveals them to be primarily immature with the latter two peaks corresponding to larger, more mature megakaryocytes.