Proteomic screening identifies PF4/Cxcl4 as a critical driver of myelofibrosis.

Despite increased understanding of the genomic landscape of Myeloproliferative Neoplasms (MPNs), the pathological mechanisms underlying abnormal megakaryocyte (Mk)-stromal crosstalk and fibrotic progression in MPNs remain unclear. We conducted mass spectrometry-based proteomics on mice with Romiplostim-dependent myelofibrosis to reveal alterations in signaling pathways and protein changes in Mks, platelets, and bone marrow (BM) cells. The chemokine Platelet Factor 4 (PF4)/Cxcl4 was up-regulated in all proteomes and increased in plasma and BM fluids of fibrotic mice. High TPO concentrations sustained in vitro PF4 synthesis and secretion in cultured Mks, while Ruxolitinib restrains the abnormal PF4 expression in vivo. We discovered that PF4 is rapidly internalized by stromal cells through surface glycosaminoglycans (GAGs) to promote myofibroblast differentiation. Cxcl4 gene silencing in Mks mitigated the profibrotic phenotype of stromal cells in TPO-saturated co-culture conditions. Consistently, extensive stromal PF4 uptake and altered GAGs deposition were detected in Romiplostim-treated, JAK2V617F mice and BM biopsies of MPN patients. BM PF4 levels and Mk/platelet CXCL4 expression were elevated in patients, exclusively in overt fibrosis. Finally, pharmacological inhibition of GAGs ameliorated in vivo fibrosis in Romiplostim-treated mice. Thus, our findings highlight the critical role of PF4 in the fibrosis progression of MPNs and substantiate the potential therapeutic strategy of neutralizing PF4-GAGs interaction.

Ascorbic acid mitigates the impact of oxidative stress in a human model of febrile seizure and mesial temporal lobe epilepsy.

Prolonged febrile seizures (FS) in children are linked to the development of temporal lobe epilepsy (MTLE). The association between these two pathologies may be ascribed to the long-term effects that FS exert on neural stem cells, negatively affecting the generation of new neurons. Among the insults associated with FS, oxidative stress is noteworthy. Here, we investigated the consequences of exposure to hydrogen peroxide (H2O2) in an induced pluripotent stem cell-derived neural stem cells (iNSCs) model of a patient affected by FS and MTLE. In our study, we compare the findings from the MTLE patient with those derived from iNSCs of a sibling exhibiting a milder phenotype defined only by FS, as well as a healthy individual. In response to H2O2 treatment, iNSCs derived from MTLE patients demonstrated an elevated production of reactive oxygen species and increased apoptosis, despite the higher expression levels of antioxidant genes and proteins compared to other cell lines analysed. Among the potential causative mechanisms of enhanced vulnerability of MTLE patient iNSCs to oxidative stress, we found that these cells express low levels of the heat shock protein HSPB1 and of the autophagy adaptor SQSTM1/p62. Pre-treatment of diseased iNSCs with the antioxidant molecule ascorbic acid restored HSBP1 and p62 expression and simultaneously reduced the levels of ROS and apoptosis. Our findings suggest the potential for rescuing the impaired oxidative stress response in diseased iNSCs through antioxidant treatment, offering a promising mechanism to prevent FS degeneration in MTLE.

Newly identified roles for PIEZO1 mechanosensor in controlling normal megakaryocyte development and in primary myelofibrosis.

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.

Lack of COL6/collagen VI causes megakaryocyte dysfunction by impairing autophagy and inducing apoptosis.

Endoplasmic reticulum stress is an emerging significant player in the molecular pathology of connective tissue disorders. In response to endoplasmic reticulum stress, cells can upregulate macroautophagy/autophagy, a fundamental cellular homeostatic process used by cells to degrade and recycle proteins or remove damaged organelles. In these scenarios, autophagy activation can support cell survival. Here we demonstrated by in vitro and in vivo approaches that megakaryocytes derived from col6a1-/- (collagen, type VI, alpha 1) null mice display increased intracellular retention of COL6 polypeptides, endoplasmic reticulum stress and apoptosis. The unfolded protein response is activated in col6a1-/- megakaryocytes, as evidenced by the upregulation of molecular chaperones, by the increased splicing of Xbp1 mRNA and by the higher level of the pro-apoptotic regulator DDIT3/CHOP. Despite the endoplasmic reticulum stress, basal autophagy is impaired in col6a1-/- megakaryocytes, which show lower BECN1 levels and reduced autophagosome maturation. Starvation and rapamycin treatment rescue the autophagic flux in col6a1-/- megakaryocytes, leading to a decrease in intracellular COL6 polypeptide retention, endoplasmic reticulum stress and apoptosis. Furthermore, megakaryocytes cultured from peripheral blood hematopoietic progenitors of patients affected by Bethlem myopathy and Ullrich congenital muscular dystrophy, two COL6-related disorders, displayed increased apoptosis, endoplasmic reticulum stress and impaired autophagy. These data demonstrate that genetic disorders of collagens, endoplasmic reticulum stress and autophagy regulation in megakaryocytes may be interrelated.Abbreviations: 7-AAD: 7-amino-actinomycin D; ATF: activating transcriptional factor; BAX: BCL2 associated X protein; BCL2: B cell leukemia/lymphoma 2; BCL2L1/Bcl-xL: BCL2-like 1; BM: bone marrow; COL6: collagen, type VI; col6a1-/-: mice that are null for Col6a1; DDIT3/CHOP/GADD153: DNA-damage inducible transcript 3; EGFP: enhanced green fluorescent protein; ER: endoplasmic reticulum; reticulophagy: endoplasmic reticulum-selective autophagy; HSPA5/Bip: heat shock protein 5; HSP90B1/GRP94: heat shock protein 90, beta (Grp94), member 1; LAMP2: lysosomal associated membrane protein 2; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; Mk: megakaryocytes; MTOR: mechanistic target of rapamycin kinase; NIMV: noninvasive mechanical ventilation; PI3K: phosphoinositide 3-kinase; PPP1R15A/GADD34: protein phosphatase 1, regulatory subunit 15A; RT-qPCR: reverse transcription-quantitative real-time PCR; ROS: reactive oxygen species; SERPINH1/HSP47: serine (or cysteine) peptidase inhibitor, clade H, member 1; sh-RNA: short hairpin RNA; SOCE: store operated calcium entry; UCMD: Ullrich congenital muscular dystrophy; UPR: unfolded protein response; WIPI2: WD repeat domain, phosphoinositide-interacting 2; WT: wild type; XBP1: X-box binding protein 1.

Reduced platelet glycoprotein Ibα shedding accelerates thrombopoiesis and COX-1 recovery: implications for aspirin dosing regimen.

Cardiovascular (CV) disease prevention with low-dose aspirin can be less effective in patients with a faster recovery of platelet (PLT) cyclooxygenase (COX)-1 activity during the 24-hour dosing interval. We previously showed that incomplete suppression of TXA2 over 24 hours can be rescued by a twice daily aspirin regimen. Here we show that reduced PLT glycoprotein (GP)Ibα shedding characterizes patients with accelerated COX-1 recovery and may contribute to higher thrombopoietin (TPO) production and higher rates of newly formed PLT, escaping aspirin inhibition over 24 hours. Two hundred aspirin-treated patients with high CV risk (100 with type 2 diabetes mellitus) were stratified according to the kinetics of PLT COX-1 activity recovery during the 10- to 24-hour dosing interval. Whole proteome analysis showed that PLT from patients with accelerated COX-1 recovery were enriched in proteins involved in cell survival, inhibition of apoptosis and cellular protrusion formation. In agreement, we documented increased plasma TPO, megakaryocyte maturation and proplatelet formation, and conversely increased PLT galactose and reduced caspase 3, phosphatidylserine exposure and ADAM17 activation, translating into diminished GPIbα cleavage and glycocalicin (GC) release. Treatment of HepG2 cells with recombinant GC led to a dose-dependent reduction of TPO mRNA in the liver, suggesting that reduced GPIbα ectodomain shedding may unleash thrombopoiesis. A cluster of clinical markers, including younger age, non-alcoholic fatty liver disease, visceral obesity and higher TPO/GC ratio, predicted with significant accuracy the likelihood of faster COX-1 recovery and suboptimal aspirin response. Circulating TPO/GC ratio, reflecting a dysregulation of PLT lifespan and production, may provide a simple tool to identify patients amenable to more frequent aspirin daily dosing.

Novel variants in GALE cause syndromic macrothrombocytopenia by disrupting glycosylation and thrombopoiesis.

Glycosylation is recognized as a key process for proper megakaryopoiesis and platelet formation. The enzyme uridine diphosphate (UDP)-galactose-4-epimerase, encoded by GALE, is involved in galactose metabolism and protein glycosylation. Here, we studied 3 patients from 2 unrelated families who showed lifelong severe thrombocytopenia, bleeding diathesis, mental retardation, mitral valve prolapse, and jaundice. Whole-exome sequencing revealed 4 variants that affect GALE, 3 of those previously unreported (Pedigree A, p.Lys78ValfsX32 and p.Thr150Met; Pedigree B, p.Val128Met; and p.Leu223Pro). Platelet phenotype analysis showed giant and/or grey platelets, impaired platelet aggregation, and severely reduced alpha and dense granule secretion. Enzymatic activity of the UDP-galactose-4-epimerase enzyme was severely decreased in all patients. Immunoblotting of platelet lysates revealed reduced GALE protein levels, a significant decrease in N-acetyl-lactosamine (LacNAc), showing a hypoglycosylation pattern, reduced surface expression of gylcoprotein Ibα-IX-V (GPIbα-IX-V) complex and mature ß1 integrin, and increased apoptosis. In vitro studies performed with patients-derived megakaryocytes showed normal ploidy and maturation but decreased proplatelet formation because of the impaired glycosylation of the GPIbα and ß1 integrin, and reduced externalization to megakaryocyte and platelet membranes. Altered distribution of filamin A and actin and delocalization of the von Willebrand factor were also shown. Overall, this study expands our knowledge of GALE-related thrombocytopenia and emphasizes the critical role of GALE in the physiological glycosylation of key proteins involved in platelet production and function.

Bone marrow microenvironment of MPN cells.

In this chapter, we will discuss the current knowledge concerning the alterations of the cellular components in the bone marrow niche in Myeloproliferative Neoplasms (MPNs), highlighting the central role of the megakaryocytes in MPN progression, and the extracellular matrix components characterizing the fibrotic bone marrow.

Ablation of collagen VI leads to the release of platelets with altered function.

Hemostatic abnormalities and impaired platelet function have been described in patients affected by connective tissue disorders. We observed a moderate bleeding tendency in patients affected by collagen VI-related disorders and investigated the defects in platelet functionality, whose mechanisms are unknown. We demonstrated that megakaryocytes express collagen VI that is involved in the regulation of functional platelet production. By exploiting a collagen VI-null mouse model (Col6a1-/-), we found that collagen VI-null platelets display significantly increased susceptibility to activation and intracellular calcium signaling. Col6a1-/- megakaryocytes and platelets showed increased expression of stromal interaction molecule 1 (STIM1) and ORAI1, the components of store-operated calcium entry (SOCE), and activation of the mammalian target of rapamycin (mTOR) signaling pathway. In vivo mTOR inhibition by rapamycin reduced STIM1 and ORAI1 expression and calcium flows, resulting in a normalization of platelet susceptibility to activation. These defects were cell autonomous, because transplantation of lineage-negative bone marrow cells from Col6a1-/- mice into lethally irradiated wild-type animals showed the same alteration in SOCE and platelet activation seen in Col6a1-/- mice. Peripheral blood platelets of patients affected by collagen VI-related diseases, Bethlem myopathy and Ullrich congenital muscular dystrophy, displayed increased expression of STIM1 and ORAI1 and were more prone to activation. Altogether, these data demonstrate the importance of collagen VI in the production of functional platelets by megakaryocytes in mouse models and in collagen VI-related diseases.

Expression and functional characterization of the large-conductance calcium and voltage-activated potassium channel Kca 1.1 in megakaryocytes and platelets.

BACKGROUND: Ion channels are transmembrane proteins that play important roles in cell function regulation modulating ionic cell permeability. In megakaryocytes and platelets, regulated ion flows have been demonstrated to modulate platelet production and function. However, a relatively limited characterization of ion channel expression and function is available in the human megakaryocyte-platelet lineage. OBJECTIVE: We analyzed the expression and function of the large-conductance calcium and voltage-activated potassium channel Kca 1.1 (also known as Maxi-K, BK, slo1) in human megakaryocytes and platelets. METHODS: To investigate the functionality of Kca 1.1, we exploited different agonists (BMS-191011, NS1619, NS11021, epoxyeicosatrienoic acid isoforms) and inhibitors (iberiotoxin, penitrem A) of the channel. RESULTS: In megakaryocytes, Kca 1.1 agonists determined a decreased proplatelet formation and altered interaction with the extracellular matrix. Analysis of the actin cytoskeleton demonstrated a significant decrease in megakaryocyte spreading and adhesion to collagen. In platelets, the opening of the channel Kca 1.1 led to a reduced sensitivity to agonists with blunted aggregation in response to ADP, with an inhibitory capacity additive to that of aspirin. The Kca 1.1 agonists, but not the inhibitors, determined a reduction of platelet adhesion and aggregation onto immobilized collagen underflow to an extent similar to that of aspirin and ticagrelor. The opening of the Kca 1.1 resulted in cell hyperpolarization impairing free intracellular calcium in ADP-stimulated platelets and megakaryocytes. CONCLUSIONS: The present study reveals new mechanisms in platelet formation and activation, suggesting that targeting Kca 1.1 channels might be of potential pharmacological interest in hemostasis and thrombosis.

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.

Mechanisms of platelet release: in vivo studies and in vitro modeling.

Mechanisms related to platelet release in the context of the bone marrow niche are not completely known. In this review we discuss what has been discovered about four critical aspects of this process: 1) the bone marrow niche organization, 2) the role of the extracellular matrix components, 3) the mechanisms by which megakaryocytes release platelets and 4) the novel approaches to mimic the bone marrow environment and produce platelets ex vivo.

Defective interaction of mutant calreticulin and SOCE in megakaryocytes from patients with myeloproliferative neoplasms.

Approximately one-fourth of patients with essential thrombocythemia or primary myelofibrosis carry a somatic mutation of the calreticulin gene (CALR), the gene encoding for calreticulin. A 52-bp deletion (type I mutation) and a 5-bp insertion (type II mutation) are the most frequent genetic lesions. The mechanism(s) by which a CALR mutation leads to a myeloproliferative phenotype has been clarified only in part. We studied the interaction between calreticulin and store-operated calcium (Ca2+) entry (SOCE) machinery in megakaryocytes (Mks) from healthy individuals and from patients with CALR-mutated myeloproliferative neoplasms (MPNs). In Mks from healthy subjects, binding of recombinant human thrombopoietin to c-Mpl induced the activation of signal transducer and activator of transcription 5, AKT, and extracellular signal-regulated kinase 1/2, determining inositol triphosphate-dependent Ca2+ release from the endoplasmic reticulum (ER). This resulted in the dissociation of the ER protein 57 (ERp57)-mediated complex between calreticulin and stromal interaction molecule 1 (STIM1), a protein of the SOCE machinery that leads to Ca2+ mobilization. In Mks from patients with CALR-mutated MPNs, defective interactions between mutant calreticulin, ERp57, and STIM1 activated SOCE and generated spontaneous cytosolic Ca2+ flows. In turn, this resulted in abnormal Mk proliferation that was reverted using a specific SOCE inhibitor. In summary, the abnormal SOCE regulation of Ca2+ flows in Mks contributes to the pathophysiology of CALR-mutated MPNs. In perspective, SOCE may represent a new therapeutic target to counteract Mk proliferation and its clinical consequences in MPNs.

Constitutive STAT5 phosphorylation in CD34+ cells of patients with primary myelofibrosis: Correlation with driver mutation status and disease severity.

Primary Myelofibrosis (PMF) is a myeloproliferative disorder associated with JAK2V617F, Calreticulin (CALR) indels, and MPLW515L/K mutations activating the tyrosine kinase JAK2 and its downstream signaling pathway. The nature of signaling abnormalities in primary cells from PMF patients is poorly understood, since most of the work has been performed in cell lines or animal models. By flow cytometry we measured constitutive and cytokine induced phosphorylation of STAT5, STAT3, and ERK1/2 in circulating CD34+ cells from 57 patients with PMF (20 with prefibrotic-PMF) and 13 healthy controls (CTRLs). Levels of constitutive and TPO induced p-STAT5, and IL6 induced p-STAT3 were higher in patients than in CTRLs. Constitutive p-STAT5 values were lower in CALR than homozygous JAK2V617F mutated CD34+ cells from PMF patients. Moreover, constitutive p-STAT5 and IL6 induced p-STAT3 values correlated directly with circulating CD34+ cell number/L, and inversely with the frequency of circulating CD34+ cells expressing CXCR4. Constitutive p-STAT5 values of CD34+ cells were also inversely correlated with hemoglobin levels. When the patients were divided according with presence/absence of JAK2V617F mutation, all the correlations described characterized the JAK2V617F+ patients with prefibrotic-PMF (P-PMF). In conclusion, increased constitutive p-STAT5 and IL6 induced p-STAT3 values in circulating CD34+ cells characterize patients with PMF. Constitutive p-STAT5 and IL6 induced p-STAT3 values correlate with circulating CD34+ cell number/L, the frequency of circulating CD34+ cells expressing CXCR4 and hemoglobin levels within the prefibrotic JAK2V617F+ patient population. Our data point toward a complex activation of STAT5-dependent pathways in the stem/progenitor cell compartment, that characterize the phenotypic diversity of PMF.

EDA fibronectin-TLR4 axis sustains megakaryocyte expansion and inflammation in bone marrow fibrosis.

The fibronectin EDA isoform (EDA FN) is instrumental in fibrogenesis but, to date, its expression and function in bone marrow (BM) fibrosis have not been explored. We found that mice constitutively expressing the EDA domain (EIIIA+/+), but not EDA knockout mice, are more prone to develop BM fibrosis upon treatment with the thrombopoietin (TPO) mimetic romiplostim (TPOhigh). Mechanistically, EDA FN binds to TLR4 and sustains progenitor cell proliferation and megakaryopoiesis in a TPO-independent fashion, inducing LPS-like responses, such as NF-κB activation and release of profibrotic IL-6. Pharmacological inhibition of TLR4 or TLR4 deletion in TPOhigh mice abrogated Mk hyperplasia, BM fibrosis, IL-6 release, extramedullary hematopoiesis, and splenomegaly. Finally, developing a novel ELISA assay, we analyzed samples from patients affected by primary myelofibrosis (PMF), a well-known pathological situation caused by altered TPO signaling, and found that the EDA FN is increased in plasma and BM biopsies of PMF patients as compared with healthy controls, correlating with fibrotic phase.

Megakaryocyte Contribution to Bone Marrow Fibrosis: many Arrows in the Quiver.

In Primary Myelofibrosis (PMF), megakaryocyte dysplasia/hyperplasia determines the release of inflammatory cytokines that, in turn, stimulate stromal cells and induce bone marrow fibrosis. The pathogenic mechanism and the cells responsible for progression to bone marrow fibrosis in PMF are not completely understood. This review article aims to provide an overview of the crucial role of megakaryocytes in myelofibrosis by discussing the role and the altered secretion of megakaryocyte-derived soluble factors, enzymes and extracellular matrices that are known to induce bone marrow fibrosis.

Three-Dimensional Tissue Models for Studying Ex Vivo Megakaryocytopoiesis and Platelet Production.

Three-dimensional (3D) tissue cultures in vitro enable a more physiological reconstruction of native tissues and organs. The bone marrow environment, structure and composition regulate megakaryocyte function and platelet production. Here, we describe the use of silk fibroin protein biomaterials to assemble 3D scaffolds mimicking the bone marrow niche architecture and extracellular matrix composition to support platelet release from human megakaryocytes. Additionally, we also propose the use of hyaluronan hydrogels, functionalized with extracellular matrix components, to reproduce the 3D matrix structure of the bone marrow environment for studying human megakaryocyte function.

The spleen of patients with myelofibrosis harbors defective mesenchymal stromal cells.

Splenic hematopoiesis is a major feature in the course of myelofibrosis (MF). In fact, the spleen of patients with MF contains malignant hematopoietic stem cells retaining a complete differentiation program, suggesting both a pivotal role of the spleen in maintaining the disease and a tight regulation of hematopoiesis by the splenic microenvironment, in particular by mesenchymal stromal cells (MSCs). Little is known about splenic MSCs (Sp-MSCs), both in normal and in pathological context. In this work, we have in vitro expanded and characterized Sp-MSCs from 25 patients with MF and 13 healthy subjects (HS). They shared similar phenotype, growth kinetics, and differentiation capacity. However, MF Sp-MSCs expressed significant lower levels of nestin, and favored megakaryocyte (Mk) differentiation in vitro at a larger extent than their normal counterpart. Moreover, they showed a significant upregulation of matrix metalloprotease 2 (MMP2) and fibronectin 1 (FN1) genes both at mRNA expression and at protein level, and, finally, developed genetic abnormalities which were never detected in HS-derived Sp-MSCs. Our data point toward the existence of a defective splenic niche in patients with MF that could be responsible of some pathological features of the disease, including the increased trafficking of CD34+ cells and the expansion of the megakaryocytic lineage.