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.

Thrombopoietin, the Primary Regulator of Platelet Production: From Mythos to Logos, a Thirty-Year Journey.

Thrombopoietin, the primary regulator of blood platelet production, was postulated to exist in 1958, but was only proven to exist when the cDNA for the hormone was cloned in 1994. Since its initial cloning and characterization, the hormone has revealed many surprises. For example, instead of acting as the postulated differentiation factor for platelet precursors, megakaryocytes, it is the most potent stimulator of megakaryocyte progenitor expansion known. Moreover, it also stimulates the survival, and in combination with stem cell factor leads to the expansion of hematopoietic stem cells. All of these growth-promoting activities have resulted in its clinical use in patients with thrombocytopenia and aplastic anemia, although the clinical development of the native molecule illustrated that "it's not wise to mess with mother nature", as a highly engineered version of the native hormone led to autoantibody formation and severe thrombocytopenia. Finally, another unexpected finding was the role of the thrombopoietin receptor in stem cell biology, including the development of myeloproliferative neoplasms, an important disorder of hematopoietic stem cells. Overall, the past 30 years of clinical and basic research has yielded many important insights, which are reviewed in this paper.

Differential in vivo roles of Mpl cytoplasmic tyrosine residues in murine hematopoiesis and myeloproliferative disease.

Thrombopoietin (Tpo), which binds to its specific receptor, the Mpl protein, is the major cytokine regulator of megakaryopoiesis and circulating platelet number. Tpo binding to Mpl triggers activation of Janus kinase 2 (Jak2) and phosphorylation of the receptor, as well as activation of several intracellular signalling cascades that mediate cellular responses. Three tyrosine (Y) residues in the C-terminal region of the Mpl intracellular domain have been implicated as sites of phosphorylation required for regulation of major Tpo-stimulated signalling pathways: Mpl-Y565, Mpl-Y599 and Mpl-Y604. Here, we have introduced mutations in the mouse germline and report a consistent physiological requirement for Mpl-Y599, mutation of which resulted in thrombocytopenia, deficient megakaryopoiesis, low hematopoietic stem cell (HSC) number and function, and attenuated responses to myelosuppression. We further show that in models of myeloproliferative neoplasms (MPN), where Mpl is required for pathogenesis, thrombocytosis was dependent on intact Mpl-Y599. In contrast, Mpl-Y565 was required for negative regulation of Tpo responses; mutation of this residue resulted in excess megakaryopoiesis at steady-state and in response to myelosuppression, and exacerbated thrombocytosis associated with MPN.

Thrombopoietin-independent generation of platelet-like particles from megakaryoblastic cells.

The use of megakaryoblastic leukemia MEG-01 cells can help reveal the mechanisms of thrombopoiesis. However, conventional in vitro activation of platelet release from MEG-01 cells requires thrombopoietin, which is costly. Here, we aim to develop a more straightforward and affordable method. Synchronization of the MEG-01 cells was initially performed using serum-free culture, followed by spontaneous cell differentiation in the presence of serum. Different stages of megakaryoblast differentiation were classified based on cell morphology, DNA content, and cell cycle. The MEG-01 cells released platelet-like particles at a level comparable to that of the thrombopoietin-activated MEG-01 cells. The platelet-like particles were distinguishable from PLP-derived extracellular vesicles and could express P-selectin following ADP activation. Importantly, the platelet-like particles induced fibrin clotting in vitro using platelet-poor plasma. Therefore, this thrombopoietin-independent cell synchronization method is an effective and straightforward method for studying megakaryopoiesis and thrombopoiesis.

Patient preferences and experiences regarding thrombopoietin-receptor agonists for immune thrombocytopenia in The Netherlands (TRAPeze Netherlands study).

OBJECTIVE: Identify patient experience and preference towards thrombopoietin-receptor agonists (TPO-RAs) in treatment of immune thrombocytopenia (ITP) in the Netherlands. METHODS: The Thrombopoietin-Receptor Agonist Patient experience (TRAPeze) survey used a discrete choice experiment (DCE) to elicit patient preferences and a patient burden survey (PBS) to evaluate the clinical and social impact of ITP. TRAPeze collected responses from 6th October to 19th November 2021. RESULTS: Seventy-six respondents completed the DCE: treatment preference appeared to be driven by method of administration (odds ratio [OR] 4.33; 95% confidence interval [CI] 2.88-6.52), frequency of dosing (OR 2.33; 95% CI 1.86-2.92) and drug-food interactions (OR 1.91; 95% CI 1.54-2.37). Respondents preferred therapies delivered orally over subcutaneous injection (OR 4.22; 95% CI 2.76-6.46), dosed once weekly over once daily (OR 2.37; 95% CI 1.58-3.54) and without food restrictions over with restrictions (OR 1.90; 95% CI 1.52-2.38). Sixty-nine respondents completed the DCE and PBS (mean [range] age 53 [19-83] years, 65% female). Seven incomplete PBS responses were excluded from analysis. Respondents were currently, or most recently, receiving eltrombopag (n = 43) or romiplostim (n = 26), of which 30% (n = 21/69) had previously received another TPO-RA. Loss (29%, n = 6/21) and lack (29%, n = 6/21) of response were the most common reasons for switching TPO-RA. Only 28% (n = 18/65) of respondents felt their TPO-RA increased energy levels. CONCLUSION: Patients preferred therapies delivered orally, dosed less frequently and without food restrictions. QoL of ITP patients on TPO-RAs can be improved; the burden analyses presented can inform future efforts towards this.

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.

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.

Insights into Regulatory Factors in Megakaryocyte Development and Function: Basic Mechanisms and Potential Targets.

Platelets are small, anucleate cellular fragments, which are produced by megakaryocytes, and play a key role in hemostasis and thrombus formation. The differentiation of megakaryocytes from hematopoietic stem cells in bone marrow and the development of megakaryocytes into platelets is a complex process. Various regulatory factorsin megakaryopoiesis including cytokines, growth factors, transcription factors, and gene expression, are all involved in the process of thrombocytopoiesis and play distinct roles in different stages of megakaryocytes development. In this review, we summarize the current state of knowledge ofmultiple regulatory factors including the TPO/Mpl signaling pathway, transcription factors, RasGTPases family, estrogen, and microRNAs. Altogether, we aimed to discuss more molecular mechanisms of megakaryocytes differentiation and maturation, and possess a better understanding of platelet formation.

c-Mpl-del, a c-Mpl alternative splicing isoform, promotes AMKL progression and chemoresistance.

Acute megakaryocytic leukemia (AMKL) is a clinically heterogeneous subtype of acute myeloid leukemia characterized by unrestricted megakaryoblast proliferation and poor prognosis. Thrombopoietin receptor c-Mpl is a primary regulator of megakaryopoeisis and a potent mitogenic receptor. Aberrant c-Mpl signaling has been implicated in a myriad of myeloid proliferative disorders, some of which can lead to AMKL, however, the role of c-Mpl in AMKL progression remains largely unexplored. Here, we identified increased expression of a c-Mpl alternative splicing isoform, c-Mpl-del, in AMKL patients. We found that c-Mpl-del expression was associated with enhanced AMKL cell proliferation and chemoresistance, and decreased survival in xenografted mice, while c-Mpl-del knockdown attenuated proliferation and restored apoptosis. Interestingly, we observed that c-Mpl-del exhibits preferential utilization of phosphorylated c-Mpl-del C-terminus Y607 and biased activation of PI3K/AKT pathway, which culminated in upregulation of GATA1 and downregulation of DDIT3-related apoptotic responses conducive to AMKL chemoresistance and proliferation. Thus, this study elucidates the critical roles of c-Mpl alternative splicing in AMKL progression and drug resistance, which may have important diagnostic and therapeutic implications for leukemia accelerated by c-Mpl-del overexpression.

Thrombopoietin participates in platelet activation in COVID-19 patients.

BACKGROUND: The pathogenesis of coronavirus disease 2019 (COVID-19) is characterized by enhanced platelet activation and diffuse hemostatic alterations, which may contribute to immunothrombosis/thromboinflammation and subsequent development of target-organ damage. Thrombopoietin (THPO), a growth factor essential to megakariocyte proliferation, is known to prime platelet activation and leukocyte-platelet interaction. In addition, THPO concentrations increase in several critical diseases, such as acute cardiac ischemia and sepsis, thus representing a potential diagnostic and prognostic biomarker. Furthermore, several data suggest that interleukin (IL)-6 is one of the most important inflammatory mediators involved in these phenomena, which led to explore the potential therapeutic role of IL-6 inhibitors. In this prospective cohort study, we aimed to study THPO and IL-6 concentrations in COVID-19 patients at the time of first clinical evaluation in the Emergency Department (ED), and to investigate their potential use as diagnostic and prognostic biomarkers. In addition, we sought to explore the role of THPO contained in plasma samples obtained from COVID-19 patients in priming in vitro platelet activation and leukocyte-platelet interaction. METHODS: We enrolled 66 patients presenting to the ED with symptoms suggestive of COVID-19, including 47 with confirmed COVID-19 and 19 in whom COVID-19 was excluded (Non-COVID-19 patients). As controls, we also recruited 18 healthy subjects. In vitro, we reproduced the effects of increased circulating THPO on platelet function by adding plasma from COVID-19 patients or controls to platelet-rich plasma or whole blood obtained by healthy donors, and we indirectly studied the effect of THPO on platelet activation by blocking its biological activity. FINDINGS: THPO levels were higher in COVID-19 patients than in both Non-COVID-19 patients and healthy subjects. Studying THPO as diagnostic marker for the diagnosis of COVID-19 by receiver-operating-characteristic (ROC) statistics, we found an area under the curve (AUC) of 0.73, with an optimal cut-off value of 42.60 pg/mL. IL-6 was higher in COVID-19 patients than in healthy subjects, but did not differ between COVID-19 and Non-COVID-19 patients. THPO concentrations measured at the time of diagnosis in the ED were also higher in COVID-19 patients subsequently developing a severe disease than in those with mild disease. Evaluating THPO as biomarker for severe COVID-19 using ROC analysis, we found an AUC of 0.71, with an optimal cut-off value of 57.11 pg/mL. IL-6 was also higher in severe than in mild COVID-19 patients, with an AUC for severe COVID-19 of 0.83 and an optimal cut-off value of 23 pg/ml. THPO concentrations correlated with those of IL-6 (r=0.2963; p=0.043), and decreased 24 h after the administration of tocilizumab, an IL-6 receptor blocking antibody, showing that the increase of THPO levels depends on IL-6-stimulated hepatic synthesis. In vitro, plasma obtained from COVID-19 patients, but not from healthy subjects, primed platelet aggregation and leukocyte-platelet binding, and these effects were reduced by inhibiting THPO activity. INTERPRETATION: Increased THPO may be proposed as an early biomarker for the diagnosis of COVID-19 and for the identification of patients at risk of developing critical illness. Elevated THPO may contribute to enhance platelet activation and leukocyte-platelet interaction in COVID-19 patients, thus potentially participating in immunothrombosis/thromboinflammation. FUNDING: This work was supported by Ministero dell'Università e della Ricerca Scientifica e Tecnologica (MURST) ex 60% to GM and EL.

Influenza A virus infection instructs hematopoiesis to megakaryocyte-lineage output.

Respiratory tract infections are among the deadliest communicable diseases worldwide. Severe cases of viral lung infections are often associated with a cytokine storm and alternating platelet numbers. We report that hematopoietic stem and progenitor cells (HSPCs) sense a non-systemic influenza A virus (IAV) infection via inflammatory cytokines. Irrespective of antiviral treatment or vaccination, at a certain threshold of IAV titer in the lung, CD41-positive hematopoietic stem cells (HSCs) enter the cell cycle while endothelial protein C receptor-positive CD41-negative HSCs remain quiescent. Active CD41-positive HSCs represent the source of megakaryocytes, while their multi-lineage reconstitution potential is reduced. This emergency megakaryopoiesis is thrombopoietin independent and attenuated in IAV-infected interleukin-1 receptor-deficient mice. Newly produced platelets during IAV infection are immature and hyper-reactive. After viral clearance, HSC quiescence is re-established. Our study reveals that non-systemic viral respiratory infection has an acute impact on HSCs via inflammatory cytokines to counteract IAV-induced thrombocytopenia.

Living Biomaterials to Engineer Hematopoietic Stem Cell Niches.

Living biointerfaces are a new class of biomaterials combining living cells and polymeric matrices that can act as biologically active and instructive materials that host and provide signals to surrounding cells. Here, living biomaterials based on Lactococcus lactis to control hematopoietic stem cells in 2D surfaces and 3D hydrogels are introduced. L. lactis is modified to express C-X-C motif chemokine ligand 12 (CXCL12), thrombopoietin (TPO), vascular cell adhesion protein 1 (VCAM1), and the 7th-10th type III domains of human plasma fibronectin (FN III7-10 ), in an attempt to mimic ex vivo the conditions of the human bone marrow. These results suggest that living biomaterials that incorporate bacteria expressing recombinant CXCL12, TPO, VCAM1, and FN in both 2D systems direct hematopoietic stem and progenitor cells (HSPCs)-bacteria interaction, and in 3D using hydrogels functionalized with full-length human plasma fibronectin allow for a notable expansion of the CD34+ /CD38- /CD90+ HSPC population compared to the initial population. These results provide a strong evidence based on data that suggest the possibility of using living materials based on genetically engineered bacteria for the ex-vivo expansion of HSPC with eventual practical clinical applications in HSPCs transplantation for hematological disorders.

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.

Generation of a thrombopoietin-deficient thrombocytopenia model in zebrafish.

BACKGROUND: The production of platelets is tightly regulated by thrombopoietin (THPO). Mutations in the THPO gene cause thrombocytopenia. Although mice lacking Thpo present with thrombocytopenia, predicting phenotypes and pathogenicity of novel THPO mutations in mice is limited. Zebrafish can be a powerful tool for fast validation and study of candidate genes of human hematological diseases and have already been used as a model of human thrombocytopenia. OBJECTIVES: We aim to investigate the role of Thpo in zebrafish thrombopoiesis and to establish a Thpo-deficient zebrafish model. The model could be applied for illustrating the clinically discovered human THPO variants of which the clinical significance is not known and to evaluate the effect of THPO receptor agonists (THPO-Ras), as well as a screening platform for new drugs. METHODS: We generated a thpo loss-of-function zebrafish model using CRISPR/Cas9. After disruption of zebrafish thpo, thposzy6 zebrafish presented with a significant reduction of thpo expression and developed thrombocytopenia. Furthermore, we performed in vivo studies with zebrafish with the thposzy6 mutation and found two human clinical point mutations (c.091C > T and c.112C > T) that were responsible for the thrombocytopenia phenotype. In addition, effects of THPO-RAs used as therapeutics against thrombocytopenia were evaluated in the Tg(mpl:eGFP);thposzy6 line. RESULTS AND CONCLUSIONS: Zebrafish with the mutation thposzy6 presented with a significant reduction of thpo expression and developed thrombocytopenia. Thpo loss-of-function zebrafish model can serve as a valuable preclinical model for thrombocytopenia caused by thpo-deficiency, as well as a tool to study human clinical THPO variants and evaluate the effect of THPO-RAs.

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.

Thrombopoietin accumulation in hepatocytes induces a decrease in its serum levels in a sinusoidal obstruction syndrome model.

Sinusoidal obstruction syndrome (SOS) is a type of fatal hepatic injury, which predominantly occurs following exposure to drugs, such as oxaliplatin, or bone marrow transplantation. Extravasated platelet aggregation (EPA) plays an important role in the development of SOS in rat and mouse models. Furthermore, platelets invading the space of Disse adhere to hepatocytes and are phagocytized in patients with SOS. Aging platelets and platelets in patients with sepsis are phagocytized by hepatocytes through Ashwell­Morell receptors, and thrombopoietin (TPO) is produced by the JAK2­STAT3 signaling pathway. The purpose of the present study was to examine the significance of TPO as a biomarker of SOS. SOS was induced in Crl:CD1(ICR) female mice by intraperitoneal administration of monocrotaline (MCT). TPO levels were measured in the serum and liver tissue. Pathological and immunohistochemical studies of the liver were performed to analyze the expression levels of TPO. TPO mRNA expression levels were measured using reverse transcription­quantitative PCR. In the SOS model, the platelet counts in peripheral blood samples were significantly decreased at 24 and 48 h after MCT treatment as compared with that at 0 h. In addition, a pathological change in hepatic zone 3 was observed in the SOS model group. Furthermore, the protein levels of TPO in liver tissue were significantly increased in the SOS model group compared with those in the control group, which was confirmed by immunohistochemistry. By contrast, serum TPO protein levels were significantly decreased in the SOS model group compared with those in the control group. These results indicated that EPA may induce sinusoidal endothelial fenestration in a mouse model of SOS, preventing TPO from translocating into the blood. In conclusion, serum TPO levels may be reduced in a mouse model of SOS owing to the accumulation in hepatocytes, suggesting that TPO could be a useful biomarker of SOS.

Megakaryocyte/platelet-derived TGF-ß1 inhibits megakaryopoiesis in bone marrow by regulating thrombopoietin production in liver.

Transforming growth factor ß1 (TGF-ß1) regulates a wide variety of events in adult bone marrow (BM), including quiescence of hematopoietic stem cells, via undefined mechanisms. Because megakaryocytes (MKs)/platelets are a rich source of TGF-ß1, we assessed whether TGF-ß1 might inhibit its own production by comparing mice with conditional inactivation of Tgfb1 in MKs (PF4Cre;Tgfb1flox/flox) and control mice. PF4Cre;Tgfb1flox/flox mice had ∼30% more MKs in BM and ∼15% more circulating platelets than control mice (P < .001). Thrombopoietin (TPO) levels in plasma and TPO expression in liver were approximately twofold higher in PF4Cre;Tgfb1flox/flox than in control mice (P < .01), whereas TPO expression in BM cells was similar between these mice. In BM cell culture, TPO treatment increased the number of MKs from wild-type mice by approximately threefold, which increased approximately twofold further in the presence of a TGF-ß1-neutralizing antibody and increased the number of MKs from PF4Cre;Tgfb1flox/flox mice approximately fourfold. Our data reveal a new role for TGF-ß1 produced by MKs/platelets in regulating its own production in BM via increased TPO production in the liver. Additional studies are required to determine the mechanism.

Hematopoietic stem cells temporally transition to thrombopoietin dependence in the fetal liver.

Tissue stem cells temporally change intrinsic mechanisms to meet physiological demands. However, little is known whether and how stem cells rely on distinct extrinsic maintenance mechanisms over time. Here, we found that hematopoietic stem cells (HSCs) temporally transition to depend on thrombopoietin (TPO), a key extrinsic factor, from E16.5 onward in the developing liver. Deletion of Tpo reduced mTOR activity, induced differentiation gene expression, and preferentially depleted metabolically active HSCs. Ectopic activation of the JAK2 or MAPK pathway did not rescue HSCs in Tpo-/- mice. Enforced activation of the mTOR pathway by conditionally deleting Tsc1 significantly rescued HSCs and their gene expression in Tpo-/- mice. Lin28b intrinsically promoted mTOR activation in HSCs, and its expression diminished over time. Conditional deletion of Lin28b further reduced mTOR activity and strongly exacerbated HSC depletion in Tpo-/- mice. Therefore, HSCs temporally transition from intrinsic LIN28B-dependent to extrinsic TPO-dependent maintenance in the developing liver.