Transition of signal requirement in hematopoietic stem cell development from hemogenic endothelial cells.

Hematopoietic stem cells (HSCs) develop from hemogenic endothelial cells (HECs) in vivo during mouse embryogenesis. When cultured in vitro, cells from the embryo phenotypically defined as pre-HSC-I and pre-HSC-II have the potential to differentiate into HSCs. However, minimal factors required for HSC induction from HECs have not yet been determined. In this study, we demonstrated that stem cell factor (SCF) and thrombopoietin (TPO) induced engrafting HSCs from embryonic day (E) 11.5 pre-HSC-I in a serum-free and feeder-free culture condition. In contrast, E10.5 pre-HSC-I and HECs required an endothelial cell layer in addition to SCF and TPO to differentiate into HSCs. A single-cell RNA sequencing analysis of E10.5 to 11.5 dorsal aortae with surrounding tissues and fetal livers detected TPO expression confined in hepatoblasts, while SCF was expressed in various tissues, including endothelial cells and hepatoblasts. Our results suggest a transition of signal requirement during HSC development from HECs. The differentiation of E10.5 HECs to E11.5 pre-HSC-I in the aorta-gonad-mesonephros region depends on SCF and endothelial cell-derived factors. Subsequently, SCF and TPO drive the differentiation of E11.5 pre-HSC-I to pre-HSC-II/HSCs in the fetal liver. The culture system established in this study provides a beneficial tool for exploring the molecular mechanisms underlying the development of HSCs from HECs.

Notch1 regulates hepatic thrombopoietin production.

ABSTRACT: Notch signaling regulates cell-fate decisions in several developmental processes and cell functions. However, the role of Notch in hepatic thrombopoietin (TPO) production remains unclear. We noted thrombocytopenia in mice with hepatic Notch1 deficiency and so investigated TPO production and other features of platelets in these mice. We found that the liver ultrastructure and hepatocyte function were comparable between control and Notch1-deficient mice. However, the Notch1-deficient mice had significantly lower plasma TPO and hepatic TPO messenger RNA levels, concomitant with lower numbers of platelets and impaired megakaryocyte differentiation and maturation, which were rescued by addition of exogenous TPO. Additionally, JAK2/STAT3 phosphorylation was significantly inhibited in Notch1-deficient hepatocytes, consistent with the RNA-sequencing analysis. JAK2/STAT3 phosphorylation and TPO production was also impaired in cultured Notch1-deficient hepatocytes after treatment with desialylated platelets. Consistently, hepatocyte-specific Notch1 deletion inhibited JAK2/STAT3 phosphorylation and hepatic TPO production induced by administration of desialylated platelets in vivo. Interestingly, Notch1 deficiency downregulated the expression of HES5 but not HES1. Moreover, desialylated platelets promoted the binding of HES5 to JAK2/STAT3, leading to JAK2/STAT3 phosphorylation and pathway activation in hepatocytes. Hepatocyte Ashwell-Morell receptor (AMR), a heterodimer of asialoglycoprotein receptor 1 [ASGR1] and ASGR2, physically associates with Notch1, and inhibition of AMR impaired Notch1 signaling activation and hepatic TPO production. Furthermore, blockage of Delta-like 4 on desialylated platelets inhibited hepatocyte Notch1 activation and HES5 expression, JAK2/STAT3 phosphorylation, and subsequent TPO production. In conclusion, our study identifies a novel regulatory role of Notch1 in hepatic TPO production, indicating that it might be a target for modulating TPO level.

Evidence for platelet-derived transforming growth factor ß1 as an early inducer of liver regeneration after hepatectomy in mice.

Platelets play a crucial role in tissue regeneration, and their involvement in liver regeneration is well-established. However, the specific contribution of platelet-derived Transforming Growth Factor Beta 1 (TGFß1) to liver regeneration remains unexplored. This study investigated the role of platelet-derived TGFß1 in initiating liver regeneration following 2/3 liver resection. Using platelet-specific TGFß1 knockout (Plt.TGFß1 KO) mice and wild-type littermates (Plt.TGFß1 WT) as controls, the study assessed circulating levels and hepatic gene expression of TGFß1, Platelet Factor 4 (PF4), and Thrombopoietin (TPO) at early time points post-hepatectomy (post-PHx). Hepatocyte proliferation was quantified through Ki67 staining and PCNA expression in total liver lysates at various intervals, and phosphohistone-H3 (PHH3) staining was employed to mark mitotic cells. Circulating levels of hepatic mitogens, Hepatocyte Growth Factor (HGF), and Interleukin-6 (IL6) were also assessed. Results revealed that platelet-TGFß1 deficiency significantly reduced total plasma TGFß1 levels at 5 h post-PHx in Plt.TGFß1 KO mice compared to controls. While circulating PF4 levels, liver platelet recruitment and activation appeared normal at early time points, Plt.TGFß1 KO mice showed more stable circulating platelet numbers with higher numbers at 48 h post-PHx. Notably, hepatocyte proliferation was significantly reduced in Plt.TGFß1 KO mice. The results show that a lack of TGFß1 in platelets leads to an unbalanced expression of IL6 in the liver and to strongly increased HGF levels 48 h after liver resection, and yet liver regeneration remains reduced. The study identifies platelet-TGFß1 as a regulator of hepatocyte proliferation and platelet homeostasis in the early stages of liver regeneration.

Thrombopoietin improves the functions of bone marrow endothelial progenitor cells via METTL16/Akt signalling of haematological patients with chemotherapy-induced thrombocytopenia.

Bone marrow endothelial progenitor cells (BM EPCs) are crucial in supporting haematopoietic regeneration, while the BM EPCs of haematological patients with chemotherapy-induced thrombocytopenia (CIT) are unavoidably damaged. Therefore, the present study aimed to examine the effect of thrombopoietin (TPO) on the recovery of BM EPCs of CIT patients and to identify the underlying mechanisms. The cell functions were determined by 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil)-acetylated low-density lipoprotein (Dil-Ac-LDL) uptake and fluorescein isothiocyanate (FITC)-labeled Ulex europaeus agglutinin-I (FITC-UEA-I) binding assay, as well as proliferation, migration and tube formation experiments. Endothelial cells were transfected with METTL16 lentivirus, followed by methylated RNA immunoprecipitation sequencing. Zebrafish with vascular defect was used as the in vivo model. TPO significantly improved the quantity and functions of BM EPCs from CIT patients in vitro and restored the subintestinal vein area of zebrafish with vascular defect in vivo. Mechanically, TPO enhanced the BM EPC functions through Akt signal mediated by METTL16, which was downregulated in BM EPCs of CIT patients and involved in the regulation of endothelial functions. The present study demonstrates that TPO improves the recovery of BM EPCs from CIT patients with haematological malignancies via METTL16/Akt signalling, which provides new insights into the role of TPO in treating CIT in addition to direct megakaryopoiesis.

Tuning MPL signaling to influence hematopoietic stem cell differentiation and inhibit essential thrombocythemia progenitors.

Thrombopoietin (TPO) and the TPO-receptor (TPO-R, or c-MPL) are essential for hematopoietic stem cell (HSC) maintenance and megakaryocyte differentiation. Agents that can modulate TPO-R signaling are highly desirable for both basic research and clinical utility. We developed a series of surrogate protein ligands for TPO-R, in the form of diabodies (DBs), that homodimerize TPO-R on the cell surface in geometries that are dictated by the DB receptor binding epitope, in effect "tuning" downstream signaling responses. These surrogate ligands exhibit diverse pharmacological properties, inducing graded signaling outputs, from full to partial TPO agonism, thus decoupling the dual functions of TPO/TPO-R. Using single-cell RNA sequencing and HSC self-renewal assays we find that partial agonistic diabodies preserved the stem-like properties of cultured HSCs, but also blocked oncogenic colony formation in essential thrombocythemia (ET) through inverse agonism. Our data suggest that dampening downstream TPO signaling is a powerful approach not only for HSC preservation in culture, but also for inhibiting oncogenic signaling through the TPO-R.

Thrombopoietin-Dependent Myelo-Megakaryopoiesis Fuels Thromboinflammation and Worsens Antibody-Mediated Chronic Renal Microvascular Injury.

SIGNIFICANCE STATEMENT: Kidney-derived thrombopoietin (TPO) increases myeloid cell and platelet production during antibody-mediated chronic kidney disease (AMCKD) in a mouse model, exacerbating chronic thromobinflammation in microvessels. The effect is mirrored in patients with extracapillary glomerulonephritis associated with thromboinflammation, TGF ß -dependent glomerulosclerosis, and increased bioavailability of TPO. Neutralization of TPO in mice normalized hematopoiesis, reduced chronic thromboinflammation, and ameliorated renal disease. The findings suggest that TPO is a relevant biomarker and a promising therapeutic target for patients with CKD and other chronic thromboinflammatory diseases.Neutralization of TPO in mice normalized hematopoiesis, reduced chronic thromboinflammation, and ameliorated renal disease. The findings suggest that TPO is a relevant biomarker and a promising therapeutic target for patients with CKD and other chronic thromboinflammatory diseases. BACKGROUND: Chronic thromboinflammation provokes microvascular alterations and rarefaction, promoting organ dysfunction in individuals with various life-threatening diseases. Hematopoietic growth factors (HGFs) released by the affected organ may sustain emergency hematopoiesis and fuel the thromboinflammatory process. METHODS: Using a murine model of antibody-mediated chronic kidney disease (AMCKD) and pharmacological interventions, we comprehensively monitored the response to injury in the circulating blood, urine, bone marrow, and kidney. RESULTS: Experimental AMCKD was associated with chronic thromboinflammation and the production of HGFs, especially thrombopoietin (TPO), by the injured kidney, which stimulated and skewed hematopoiesis toward myelo-megakaryopoiesis. AMCKD was characterized by vascular and kidney dysfunction, TGF ß -dependent glomerulosclerosis, and microvascular rarefaction. In humans, extracapillary glomerulonephritis is associated with thromboinflammation, TGF ß -dependent glomerulosclerosis, and increased bioavailability of TPO. Analysis of albumin, HGF, and inflammatory cytokine levels in sera from patients with extracapillary glomerulonephritis allowed us to identify treatment responders. Strikingly, TPO neutralization in the experimental AMCKD model normalized hematopoiesis, reduced chronic thromboinflammation, and ameliorated renal disease. CONCLUSION: TPO-skewed hematopoiesis exacerbates chronic thromboinflammation in microvessels and worsens AMCKD. TPO is both a relevant biomarker and a promising therapeutic target in humans with CKD and other chronic thromboinflammatory diseases.

[Thrombopoietin-mediated regulation of hematopoietic stem cells].

Sustaining lifelong hematopoiesis requires maintenance, proliferation, and differentiation of hematopoietic stem cells. Thrombopoietin is a cytokine essential for regulation of hematopoietic stem cells as well as differentiation and maturation of megakaryocytes required for platelet production. Due to these properties, thrombopoietin agonists have been used to treat bone marrow failure syndromes such as aplastic anemia. Through analysis of thrombopoietin gene-deficient mice, my colleagues and I have demonstrated the mechanism of action of thrombopoietin receptor agonists in hematopoietic stem cell maintenance and differentiation. This review focuses on governance of homeostasis in the hematopoietic system by thrombopoietin signaling.

Increased B4GALT1 expression is associated with platelet surface galactosylation and thrombopoietin plasma levels in MPNs.

Aberrant megakaryopoiesis is a hallmark of the myeloproliferative neoplasms (MPNs), a group of clonal hematological malignancies originating from hematopoietic stem cells, leading to an increase in mature blood cells in the peripheral blood. Sialylated derivatives of the glycan structure ß4-N-acetyllactosamine (Galß1,4GlcNAc or type-2 LacNAc, hereafter referred to as LacNAc) regulate platelet life span, hepatic thrombopoietin (TPO) production, and thrombopoiesis. We found increased TPO plasma levels in MPNs with high allele burden of the mutated clones. Remarkably, platelets isolated from MPNs had a significant increase in LacNAc expression that correlated with the high allele burden regardless of the underlying identified mutation. Megakaryocytes derived in vitro from these patients showed an increased expression of the B4GALT1 gene encoding ß-1,4-galactosyltransferase 1 (ß4GalT1). Consistently, megakaryocytes from MPN showed increased LacNAc expression relative to healthy controls, which was counteracted by the treatment with a Janus kinase 1/2 inhibitor. Altered expression of B4GALT1 in mutant megakaryocytes can lead to the production of platelets with aberrant galactosylation, which in turn promote hepatic TPO synthesis regardless of platelet mass. Our findings provide a new paradigm for understanding aberrant megakaryopoiesis in MPNs and identify ß4GalT1 as a potential actionable target for therapy.

In chronic lymphocytic leukemia, activation of the thrombopoietin receptor promotes T-cell inhibitory properties, contributing to immunosuppression.

In chronic lymphocytic leukemia (CLL), the immune system is skewed towards a suppressive milieu. Levels of thrombopoietin (TPO), promoting cellular immune regulatory activity in immune thrombocytopenic purpura, were shown to be elevated in CLL patients. This study explored TPO as a potential immunomodulator, supporting CLL progression. We evaluated CLL cell-induced expression of TPO receptor (TPO-R) on T-cells and effects of its activation on T-cell responses. CLL cell involvement in TPO generation was also assessed. Baseline TPO-R expression on CD4 + T-cells was found to be higher in CLL patients than in healthy controls (HC). Exposure of HC-T-cells to B-cells, especially to CLL-B-cells stimulated with B-cell activating molecules, resulted in enhanced TPO-R expression on T-cells. CLL-T-cell stimulation with TPO reduced their proliferation and expanded the regulatory T-cell (Treg) population. At baseline, phosphorylation of STAT5, known to impact the Treg phenotype, was elevated in CLL-T-cells relative to those of HC. Exposure to TPO further enhanced STAT5 phosphorylation in CLL-T-cells, possibly driving the observed Treg expansion. The CLL immune milieu is involved in promotion of inhibitory features in T-cells through increased TPO-R levels and TPO-induced intracellular signaling. TPO and its signaling pathway could potentially support immunosuppression in CLL, and may emerge as novel therapeutic targets.

Thrombopoietin-based CAR-T cells demonstrate in vitro and in vivo cytotoxicity to MPL positive acute myelogenous leukemia and hematopoietic stem cells.

While targeting CD19+ hematologic malignancies with CAR T cell therapy using single chain variable fragments (scFv) has been highly successful, novel strategies for applying CAR T cell therapy with other tumor types are necessary. In the current study, CAR T cells were designed using a ligand binding domain instead of an scFv to target stem-like leukemia cells. Thrombopoietin (TPO), the natural ligand to the myeloproliferative leukemia protein (MPL) receptor, was used as the antigen binding domain to engage MPL expressed on hematopoietic stem cells (HSC) and erythropoietic and megakaryocytic acute myeloid leukemias (AML). TPO-CAR T cells were tested in vitro against AML cell lines with varied MPL expression to test specificity. TPO-CAR T cells were specifically activating and cytotoxic against MPL+ leukemia cell lines. Though the TPO-CAR T cells did not extend survival in vivo, it successfully cleared the MPL+ fraction of leukemia cells. As expected, we also show the TPO-CAR is cytotoxic against MPL expressing bone marrow compartment in AML xenograft models. The data collected demonstrate preclinical potential of TPO-CAR T cells for stem-like leukemia through assessment of targeted killing of MPL+ cells and may facilitate subsequent HSC transplant under reduced intensity conditioning regimens.

Discovery of a novel megakaryopoiesis enhancer, ingenol, promoting thrombopoiesis through PI3K-Akt signaling independent of thrombopoietin.

Thrombocytopenia, a most common complication of radiotherapy and chemotherapy, is an important cause of morbidity and mortality in cancer patients. However, there are still no approved agents for the treatment of radiation- and chemotherapy-induced thrombocytopenia (RIT and CIT, respectively). In this study, a drug screening model for predicting compounds with activity in promoting megakaryocyte (MK) differentiation and platelet production was established based on machine learning (ML), and a natural product ingenol was predicted as a potential active compound. Then, in vitro experiments showed that ingenol significantly promoted MK differentiation in K562 and HEL cells. Furthermore, a RIT mice model and c-MPL knock-out (c-MPL-/-) mice constructed by CRISPR/Cas9 technology were used to assess the therapeutic action of ingenol on thrombocytopenia. The results showed that ingenol accelerated megakaryopoiesis and thrombopoiesis both in RIT mice and c-MPL-/- mice. Next, RNA-sequencing (RNA-seq) was carried out to analyze the gene expression profile induced by ingenol during MK differentiation. Finally, through experimental verifications, we demonstrated that the activation of PI3K/Akt signaling pathway was involved in ingenol-induced MK differentiation. Blocking PI3K/Akt signaling pathway abolished the promotion of ingenol on MK differentiation. Nevertheless, inhibition of TPO/c-MPL signaling pathway could not suppress ingenol-induced MK differentiation. In conclusion, our study builds a drug screening model to discover active compounds against thrombocytopenia, reveals the critical roles of ingenol in promoting MK differentiation and platelet production, and provides a promising avenue for the treatment of RIT.

Carboplatin-Induced Thrombocytopenia through JAK2 Downregulation, S-Phase Cell Cycle Arrest and Apoptosis in Megakaryocytes.

Chemotherapy-induced thrombocytopenia (CIT) is a common complication when treating malignancies with cytotoxic agents wherein carboplatin is one of the most typical agents causing CIT. Janus kinase 2 (JAK2) is one of the critical enzymes to megakaryocyte proliferation and differentiation. However, the role of the JAK2 in CIT remains unclear. In this study, we used both carboplatin-induced CIT mice and MEG-01 cell line to examine the expression of JAK2 and signal transducer and activator of transcription 3 (STAT3) pathway. Under CIT, the expression of JAK2 was significantly reduced in vivo and in vitro. More surprisingly, the JAK2/STAT3 pathway remained inactivated even when thrombopoietin (TPO) was administered. On the other hand, carboplatin could cause prominent S phase cell cycle arrest and markedly increased apoptosis in MEG-01 cells. These results showed that the thrombopoiesis might be interfered through the downregulation of JAK2/STAT3 pathway by carboplatin in CIT, and the fact that exogenous TPO supplement cannot reactivate this pathway.

miR-486-5p and miR-22-3p Enable Megakaryocytic Differentiation of Hematopoietic Stem and Progenitor Cells without Thrombopoietin.

Megakaryocytes release submicron size microparticles (MkMPs) in circulation. We have shown that MkMPs target CD34+ hematopoietic stem/progenitor cells (HSPCs) to induce megakaryocytic differentiation, and that small RNAs in MkMPs play an important role in the development of this phenotype. Here, using single-molecule real-time (SMRT) RNA sequencing (RNAseq), we identify the synergetic effect of two microRNAs (miRs), miR-486-5p and miR-22-3p (highly enriched in MkMPs), in driving the Mk differentiation of HSPCs in the absence of thrombopoietin (TPO). Separately, our data suggest that the MkMP-induced Mk differentiation of HSPCs is enabled through JNK and PI3K/Akt/mTOR signaling. The interaction between the two signaling pathways is likely mediated by a direct target of miR-486-5p and a negative regulator of PI3K/Akt signaling, the phosphatase and tensin homologue (PTEN) protein. Our data provide a possible mechanistic explanation of the biological effect of MkMPs in inducing megakaryocytic differentiation of HSPCs, a phenotype of potential physiological significance in stress megakaryopoiesis.

In and out: Traffic and dynamics of thrombopoietin receptor.

Thrombopoiesis had long been a challenging area of study due to the rarity of megakaryocyte precursors in the bone marrow and the incomplete understanding of its regulatory cytokines. A breakthrough was achieved in the early 1990s with the discovery of the thrombopoietin receptor (TpoR) and its ligand thrombopoietin (TPO). This accelerated research in thrombopoiesis, including the uncovering of the molecular basis of myeloproliferative neoplasms (MPN) and the advent of drugs to treat thrombocytopenic purpura. TpoR mutations affecting its membrane dynamics or transport were increasingly associated with pathologies such as MPN and thrombocytosis. It also became apparent that TpoR affected hematopoietic stem cell (HSC) quiescence while priming hematopoietic stem cells (HSCs) towards the megakaryocyte lineage. Thorough knowledge of TpoR surface localization, dimerization, dynamics and stability is therefore crucial to understanding thrombopoiesis and related pathologies. In this review, we will discuss the mechanisms of TpoR traffic. We will focus on the recent progress in TpoR membrane dynamics and highlight the areas that remain unexplored.

ELF3-AS1 contributes to gastric cancer progression by binding to hnRNPK and induces thrombocytosis in peripheral blood.

Numerous studies have reported that a variety of long noncoding RNAs (lncRNAs) can promote the proliferation, invasion, and migration of different tumor cells. However, different lncRNAs regulate cell functions in various forms, and the exact mechanisms are not clear. Here, we investigated the effect of the lncRNA ELF3-AS1 on gastric cancer (GC) cell function and explored the exact mechanism. Quantitative real-time polymerase chain reaction was used to detect the expression of ELF3-AS1 in GC tissues and adjacent nontumor tissues. Knockdown and overexpression of ELF3-AS1 was used to detect the effect of ELF3-AS1 on cell function. Potential downstream target genes were identified using RNA transcriptome sequencing, while RNA immunoprecipitation, chromatin immunoprecipitation, and Western blotting were performed to explore the tumor promotion mechanisms of ELF3-AS1. We observed that ELF3-AS1 was highly expressed in GC tissues, and high ELF3-AS1 expression predicted poor prognosis. The knockdown of ELF3-AS1 significantly inhibited cell proliferation, migration, and epithelial-mesenchymal transition and promoted apoptosis. Mechanistic investigations revealed that ELF3-AS1 may regulate the downstream target gene, C-C motif chemokine 20, by binding with the RNA-binding protein hnRNPK. Additionally, we found that high ELF3-AS1 expression was associated with thrombocytosis. Interleukin-6 and thrombopoietin may be involved in ELF3-AS1-induced paraneoplastic thrombocytosis. Together, our results demonstrate that aberrantly expressed ELF3-AS1 in GC may play important roles in oncogenesis and progression and is expected to become a new target for the diagnosis and treatment of GC.

Eltrombopag maintains human hematopoietic stem and progenitor cells under inflammatory conditions mediated by IFN-γ.

The proinflammatory cytokine interferon-γ (IFN-γ) has been implicated in human hematopoietic stem and progenitor cell (HSPC) depletion in immune-mediated bone marrow failure syndromes. We show that IFN-γ specifically prevents full engagement of thrombopoietin (TPO), a primary positive regulator of HSPC survival, to its receptor (c-MPL) via steric occlusion of the low-affinity binding site, contributing to perturbation of TPO-induced signaling pathways and decreased survival of human HSPCs. Eltrombopag, a synthetic small molecule mimetic of TPO that interacts with c-MPL at a position distinct from the extracellular binding site of TPO, bypasses this inhibition, providing an explanation for its clinical activity in bone marrow failure, despite already elevated endogenous TPO levels. Thus, IFN-γ-mediated perturbation of TPO:c-MPL complex formation and the resulting inhibition of a critical pathway of growth factor cell signaling may represent a general mechanism by which IFN-γ impairs the function of human HSPCs. This understanding could have broad therapeutic implications for various disorders of chronic inflammation.

Clinical applications of thrombopoietin silencing: A possible therapeutic role in COVID-19?

Thrombopoietin (TPO) is most recognized for its function as the primary regulator of megakaryocyte (MK) expansion and differentiation. MKs, in turn, are best known for their role in platelet production. Research indicates that MKs and platelets play an extensive role in the pathologic thrombosis at sites of high inflammation. TPO, therefore, is a key mediator of thromboinflammation. Silencing of TPO has been shown to decrease platelets levels and rates of pathologic thrombosis in patients with various inflammatory disorders (Barrett et al, 2020; Bunting et al, 1997; Desai et al, 2018; Kaser et al, 2001; Shirai et al, 2019). Given the high rates of thromboinflammmation in the novel coronavirus 2019 (COVID-19), as well as the well-documented aberrant MK activity in affected patients, TPO silencing offers a potential therapeutic modality in the treatment of COVID-19 and other pathologies associated with thromboinflammation. The current review explores the current clinical applications of TPO silencing and offers insight into a potential role in the treatment of COVID-19.

Evaluation of angiogenic signaling molecules associated with reactive thrombocytosis in an iron-deficient rat model.

BACKGROUND: Iron deficiency anemia (IDA)-induced reactive thrombocytosis can occur in children and adults. The underlying mechanism for this phenomenon is indeterminate. Traditional cytokines such as thrombopoietin (TPO), interleukin-6 (IL-6), and IL-11 involved in megakaryopoiesis have not been shown to be the cause. Recent studies suggest that growth factors and signaling molecules involved with angiogenesis influence the proliferation and differentiation of megakaryocytes. METHODS: We investigated the possible association between angiogenic cytokines with reactive thrombocytosis due to IDA in an iron-deficient (ID) rat model. Complete blood count, iron panels, and TPO levels were measured at baseline and 5 weeks later in both control (C) and ID rats. Angiogenic cytokines were evaluated in the bone marrow in all rats. RESULTS: We successfully induced IDA in our rats by phlebotomy and reduced iron diet. We did not find an increase of TPO in ID rats. A review of the bone marrow showed an increase in the number of megakaryocytes, vascular structures, as well as increased intensity of stain for vascular endothelial growth factor (VEGF), and CXC chemokine receptor 4 (CXCR4) in rats with IDA compared to controls. CONCLUSIONS: Our results of histological bone marrow data suggest an important role for angiogenesis in the development of IDA-induced thrombocytosis. IMPACT: Thrombocytosis is common with IDA in both children and adults, but the mechanism is unclear. We confirmed that TPO is not the major driver of iron deficiency-associated thrombocytosis. We confirmed the increase in the number of megakaryocytes in the bone marrow despite stable TPO levels. We provided evidence supporting an important role of angiogenesis in megakaryocytopoiesis/thrombopoiesis with increased vascular structures and angiogenic cytokines in the bone marrow of iron-deficient rats. The demonstration that angiogenesis may play an important role in secondary thrombocytosis could lead to a new approach in treating symptomatic reactive thrombocytosis by targeting angiogenesis.

Quantifying single-cell secretion in real time using resonant hyperspectral imaging.

Cell communication is primarily regulated by secreted proteins, whose inhomogeneous secretion often indicates physiological disorder. Parallel monitoring of innate protein-secretion kinetics from individual cells is thus crucial to unravel systemic malfunctions. Here, we report a label-free, high-throughput method for parallel, in vitro, and real-time analysis of specific single-cell signaling using hyperspectral photonic crystal resonant technology. Heterogeneity in physiological thrombopoietin expression from individual HepG2 liver cells in response to platelet desialylation was quantified demonstrating how mapping real-time protein secretion can provide a simple, yet powerful approach for studying complex physiological systems regulating protein production at single-cell resolution.