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

Alessandro Malara^1-2, Vittorio Abbonante^1-2, Maria Zingariello³, Annarita Migliaccio⁴ and Alessandra Balduini^1,2,5.

¹ Department of Molecular Medicine, University of Pavia, Pavia, Italy.
² Biotechnology Research Laboratories, IRCCS San Matteo Foundation, Pavia, Italy.
³ Unit of Microscopic and Ultrastructural Anatomy, Department of Medicine, University Campus Bio-Medico, Rome, Italy
⁴ Department of Biomedical and Neuromotorial Sciences, Alma Mater University, Bologna, Italy.
⁵ Department of Biomedical Engineering, Tufts University, Medford, MA, USA

Introduction

Megakaryocyte cargo in physiological thrombopoiesis

How do megakaryocytes target and deliver their protein cargo within bone marrow milieu? 

The constitutive in vitro secretion of both α- and dense granules-derived bioactive molecules by developing Mks has been previously demonstrated.[31,32] These functional experiments revealed that secretion of intracellular Mk products governs autocrine mechanisms that sustain cell development and platelet release.[33-36] On the contrary, the physiological in vivo relevance of these autocrine loops has been less explored. However, recent data shed new light on the in vivo involvement of Mks in maintaining BM homeostasis though the controlled release of targeted stimuli.[37] To this regard, conditional ablation of Mks in mice resulted in increased BM HSC frequency and cycling, suggesting that Mks normally restrain HSC proliferation through the production of CXCL4 and TGF-β.[13,14] Conversely, Mk production of fibroblast growth factor-1 (FGF-1) is thought to play a key role in supporting HSC and osteoblast expansion recovery following myeloablative therapy.[16] Therefore, these studies raise the fundamental question: how is Mk compound exocytosis regulated during physiologically or pathologically BM functions? Differently from Mks, platelet activation is at the heart of the control of vascular integrity.[38] During circulation, platelets are reactive to various stimuli and release the materials stored in their specific granules.[39] The extrusion of storage granules' content to the platelet's environment occurs according to regulated secretion events: movements of granules, apposition and fusion of granules and plasma membranes.[40] This 'release reaction' is a key step of primary hemostasis, but it participates also in inflammation, atherosclerosis, antimicrobial host defense, wound healing, angiogenesis, and malignancy.[41] Our current understanding of Mk/platelet secretion at the molecular level is still insufficient to explain how the careful balance between all the bioactive molecules released from granules, under certain activation conditions, is achieved. One of the hypotheses is that Mk/platelet granules are not uniform and may be differentially packaged and thus released in a segregated manner following specific stimuli.[42]
To this regard, Ma et al. observed that platelet stimulation with specific protease-activated receptor- 1 (PAR-1) or PAR-4 agonist resulted in the preferential release of VEGF or endostatin (anti- and pro-angiogenic factors, respectively).[24,43] More recently, a super-resolution immunofluorescence co-localization analysis of 15 platelet α-granule cargoes failed to confirm any functional co- clustering of these proteins.[44] Moreover, Zingariello et al. demonstrated by immunoelectron microscopy that P-selectin and von Willebrand factor (vWF) are co-localized within the same intracellular α-granules in immature Mks. The two proteins, however, are not co-localized in α- granules of mature Mks after wild type mice treatment with TPO.[45] These results suggested that P- selectin and vWF are associated in the Mk cytoplasm at early stages of maturation but that they are routed into separate anti-angiogenic or pro-angiogenic α-granule subtypes as these cells mature. Thus, the differential association of vWF and P-selectin with anti-angiogenic or pro-angiogenic factors suggest that a mechanism regulating the sequential release of different α-granule subtypes may be involved in Mk/platelet function during tissue repair.[45] Interestingly, Mks derived from the GATA-1^low mice, which harbors a hypomorphic mutation that blocks Mk maturation and displays BM fibrosis, showed reduced levels of expression of vWF and displaced P-selectin on the demarcation membrane system.
Further, the loss of alpha granules within BM Mks in a mouse model of Gray Platelet Syndrome (GPS) induced a myelofibrotic phenotype.[46] In PMF patients, ultrastructural abnormalities and variety in Mk-granules were reported by Thiele et al. more than 25 years ago.[47] Platelets derived from MPN patients showed several qualitative abnormalities, including decreased alpha granules and mitochondria and also alterations of the dense and tubular canalicular system.[48] Reduced levels of ADT, ATP and serotonin content in dense granules and lower content of beta-thromboglobulin (BTG) and platelet factor 4 (PF4) in alpha granules were also described.[49,50] Thus, aberrant assembly and secretion of Mk granules represent a potential mechanism of BM fibrosis progression (Figure 1).

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Figure 1. Schematic representation of potential mechanisms for aberrant release of Mk content in bone marrow fibrosis.

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A second intriguingly hypothesis is that intracellular Mk/platelet products may be delivered in the surrounding space by extracellular vesicles (EVs). EVs are membrane-enclosed structures of varying size (50-10,000 nm) released from cells to mediate both local and distant intercellular communication.[51] EVs of various shapes and sizes have been demonstrated in several body fluids, with substantial variation in their structure, content and function.[52] There are three main types: exosomes (50-100 nm), microparticles (200 nm-1 µm) and apoptotic bodies. Protein, lipid and RNA components contribute to cell-cell crosstalk at a short distance, in a paracrine or endocrine manner via the bloodstream. In addition, they may transfer surface receptors from one cell to another and deliver proteins, mRNA, bioactive lipids, and even whole organelles (e.g., mitochondria) into target cells.[53] It has been reported that two mechanisms used by target cells to integrate EVs are cell endocytosis and membrane fusion.[53] Recent studies of EVs in the BM have shown that these vesicles serve to regulate hematopoiesis, participate in immune cell activation, and hemostasis.[54,55] Several lines of evidence suggest that EVs are involved in regulating BM function during homeostasis and in response to injury, but also that hematological malignancies such as leukemia, multiple myeloma or viral infections can exploit EVs trafficking to reinforce tumor growth, chemotherapeutic resistance, invasion and metastasis.[56,57] Platelet-derived vesicles were first identified by electron microscopy over 50 years ago, but the definition of their features and activities have only become a major focus of interest in recent years.[58] Platelet-derived microparticles (PMPs) are released from the platelet surface and are distinguished from platelet exosomes, which are derived from endocytosis and released from multivesicular endosomes.[59] PMPs may directly stimulate other cells (e.g., hematopoietic cells, lymphocytes and endothelium),[60] transfer platelet expressed receptors (e.g., CD41 or CXCR4) to the surface of other cells,[61] and, in some situations, transfer mRNA, proteins, and even infectious particles to the target cells. Interestingly, in healthy donors, the majority of circulating CD41+ PMPs do not express surface activation marker CD62P, suggesting that they do not originate from activated platelets.[62] In a very elegant study, Flaumenhaft et al. report that a significant number of circulating CD41+ MPs in healthy individuals are derived directly from Mks.[63] Authors first demonstrated via electron microscopy of spontaneous formation of Mk-derived MPs (MkMPs) from cultured murine Mk and that these MkMPs were different from PMPs. However, a functional role for MkMPs was not revealed until a recent study, which documented a novel biological role of MkMPs that are able to induce HSC differentiation towards the Mk lineage without exogenous TPO stimulation.[64] In this paper, Jiang et al. demonstrated that MkMPs, which are distinct from Mk exosomes, target HSC with high specificity since they have no effect on other BM-resident cells, such as mesenchymal stem cells, endothelial cells or granulocytes. They showed that both endocytosis and membrane fusion were responsible for the delivery of MkMP cargo to HSCs, and that MkMPs attached to and entered HSCs preferentially through their uropods, with CD54, CD11b, CD18 and CD43 being involved in target-cell recognition.[64] Aside from the role of EVs in the Mk-HSC crosstalk in the BM under physiological conditions, we can speculate that EV trafficking may also play a distinct role in deregulated hematopoiesis during fibrotic progression (Figure 1). Interestingly, increased MP generation under high shear stress has been reported in platelets in the presence of TPO,[65] a main trigger of BM fibrosis in human and mice.

Megakaryocyte-derived pro-fibrotic cytokines in bone marrow fibrosis

In MPNs different mutations lead to myelofibrosis. The most frequent driver mutation in MPNs, JAK2 V617F, is found in 50-60% of PMF as well as 50-60% of ET, but in almost all cases of PV.[66] It is now clear that the clinical phenotype of myelofibrosis is a consequence both of primary clonal myeloproliferation and secondary inflammation, characterized by profound changes to BM stromal compartment and an atypical cytokine storm.[67] Several evidences argue for an impaired microenvironment in association with inflammation rather than one single genetic trigger: 1) MPNs (PV, ET, and PMF) are all characterized by a significant change in the cytokine production mirrored by increased plasma levels of several inflammatory cytokines (e.g., IL1, IL2, IL6, IL8, IL12, TNFα, and IFNγ), growth factors (e.g., GM-CSF, G-CSF, HGF, PDGF, and EGF), and  angiogenic factors (e.g., VEGF);[68] 2) clinical evidences that chronic inflammation is responsible of the constitutional symptoms which negatively affect the quality of life of MPN patients;[69] 3) clinical use of JAK inhibitors has confirmed that functional symptoms and splenomegaly in patients were concomitant  with a significant increase in the plasma levels of many cytokines.[70]

In PMF, Mks and monocytes are supposed to be the main source of reactive cytokines that force fibroblast proliferation, fibrotic evolution, neoangiogenesis, and osteosclerosis.[11] Several lines of evidence obtained both from studies of patients or murine models are in favor of a crucial role of Mk in myelofibrosis induction: 1) all driver mutations in MPNs result in overproduction of abnormal Mks by hyperactive Jak2/Stat signaling;[71] 2) mice bearing a human JAK2V617F gene restricted exclusively to the Mk lineage develop many of the features of MPNs;[72] 3) patients and rats treated with TPO mimetics show BM fibrosis (usually reversible);[73] 4) high and persistent TPO production by transduced hematopoietic cells in mice results in a fatal myeloproliferative disorder that has a number of features in common with human PMF;[74] 5) impaired expression of the transcription factor GATA-1, involved in erythroid and megakaryocytic differentiation, results in the development of myelofibrosis;[75] 6) patients with GPS, a rare macro- thrombocytopenia with agranular Mk/platelets, manifest myelofibrosis and splenomegaly.[76] On the contrary, contribution of monocytes is less clear. Monocytes from patients with PMF were reported to be spontaneously activated and to secrete abnormally TGF-β1,[77] however, TPO overexpression inducing BM fibrosis in NOD/SCID mice, which harbor impaired mononuclear phagocyte functions, led to controversial data.[78,79] Thus, based on the potential role of inflammation during fibrotic progression, we below describe the involvement and features of individual main Mk- derived cytokines/chemokines in the context of myelofibrosis (Table 1).

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Table 1. List of Mk-derived cytokines implicated in bone marrow fibrosis progression.

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Platelet-Derived Growth Factor (PDGF). PDGF is one of the first growth factors that has been implicated in the role of Mk in development of BM fibrosis.[80] PDGF is produced by Mks in the BM and is physiologically carried to the circulation in the α-granules of the platelets to act at the site of tissue injury as a mediator of tissue repair.[81] PDGF receptors (PDGFRs) are members of the membrane tyrosin-kinase family, composed of the two subunits PDGFRα and PDGFRβ, which form homo- or heterodimers. In the context of tissue repair, the PDGF/PDGFR axis not only enhances the replication, survival, and migration of myofibroblasts but also modulates the production and release of several pro- and anti-inflammatory mediators in fibrotic diseases.[82] Interestingly, PDGF has been reported to increase the expression of the collagen cross-linking enzyme, Lysil Oxidase (LOX), which in turn, oxidizes the PDGF receptor on smooth muscle cells, fibroblasts, and Mks, enhancing the proliferation signaling from this cytokine.[83,84] Ultimately, this loop has the potential to further boost the fibrotic phenotype.[85]
Increased levels of PDGF in plasma and urine from patients with MPNs have been reported.[86,87] Further, Mks and erythroid precursors contained increased levels of immunohistochemically detectable PDGF in BM biopsies of PMF patients.[88] The expression of members of the PDGF system in BM cells derived from PMF patients has been also investigated by real-time RT-PCR.
Increased expression of PDGFs could be demonstrated to be a feature of advanced fibrosis in PMF that is not demonstrable in the pre-fibrotic phase of the disease.[88]
Differently from their ligands, up-regulation of both PDGFRs during fibrotic progression is more controversial. In normal BM PDGFRα appeared in endothelial and endosteal cells in addition to strong labeling in Mks and platelets. In contrast, PDGFRβ subunit marked perisinusoidal stromal cells and adventitial fibrocytes of the larger vessels. However, in PMF patients, Bedekovics et al. found that PDGFRβ expression closely correlates with the grade of MF, while this was not evident for PDGFRα.[90] On the contrary, Bock et al. reported a strong up-regulation of the PDGFRα in patients with advanced myelofibrosis.[89]
Recently, the involvement of the PDGF/PDGFR axis in BM fibrosis has been definitively proven. Conditional deletion of the PDGFR-α gene and inhibition of PDGFRα by imatinib in leptin receptor+ stromal cells was shown to suppress their expansion and to ameliorate BM fibrosis in mice.[91]
Transforming Growth Factor-β (TGF-β). Among the abnormally expressed cytokines in PMF, TGF-β1 has received attention due to its critical role in inducing fibrosis not only in BM, but also in other organs.[92] TGF-β occurs in 3 isoforms: TGFβ1, TGFβ2 and TGFβ3. TGFβ1 is the most abundant of all these isoforms and platelets, Mks and monocytes cells are sources of TGF-β production.[93] TGFβ1 is secreted as latent protein and is stored in the extracellular matrix. Reactive oxygen species, proteases, integrins and thrombospondin-1 (TSP-1), convert the inactive latent complexes to the active forms.
Once activated, TGFβ-1 induces BM fibrosis on one hand, by increasing the synthesis of types I, III and IV collagen, FN, proteoglycans and tenascin;[94] while on the other hand, by decreasing matrix degradation through down-regulation of metalloproteinases (MMPs), particularly MMP3, and up- regulation of tissue inhibitors of metalloproteinase (TIMP), particularly TIMP-1.[95] However, effects of TGF-β1 are not restricted to the stromal compartment and TGF-β1-mediated changes to the BM niche remain to be fully elucidated. It is well known that TGF-β has direct effects on hematopoietic cells by negatively regulating granulocyte, erythroid, Mk and macrophage progenitor proliferation.[96] Further, Erba et al. showed that release and activation of TGF-β1 by Mks and platelets, forced endothelial cells from the BM microvasculature of PMF patients, and mouse model of PMF, to acquire a mesenchymal phenotype through Endothelial Mesenchymal Transition (EndMT), during the development of fibrosis.[97]
Not surprisingly, several groups reported on quantitative alterations of TGF-β and its receptors in Mk, platelets, and CD34+ progenitor cells from MPN patients and concluded that TGF-β was involved in myelofibrosis and myeloproliferation.[31,98-100] In addition to quantitative alterations, Ciaffoni et al., recently demonstrated abnormalities in TGF-β1 signaling genes in the marrow and spleen of PMF patients.[101] These alterations included genes of TGF-β1 signaling, cell cycling, Hedgehog and p53 signaling and suggested a non-canonical TGF-β1 signaling in marrow identifying, for the first time, autoimmunity as a possible cause of BM fibrosis in PMF.[101] Among the genes that predict the activation of the non-canonical TGF-β signaling, expression level of the Jun gene was increased in BM of PMF patients. Interestingly, over-expression of Jun was sufficient to induce myelofibrosis, severe fibrosis in multiple organs and steatohepatosis in mice.[102]
Moreover, the involvement of TGF-β in in vivo mouse model has been deeply investigated. TGF-β was significantly increased in the extracellular fluid of the BM, plasma and platelet extracts in two widely MF studied mouse models, which include the TPO^high and the GATA-1^low mice.[103,104] To test directly the impact of TGF-β1 in the pathogenesis of MF, BM stem cells from homozygous TGF-β1 null (TGF-β1(-/-)) and wild-type littermates were infected with a retrovirus encoding the murine TPO protein and engrafted into lethally irradiated wild type hosts for long-term reconstitution. Differently from wild type mice, none of the mice repopulated with TGF-β1(-/-) cells showed deposition of reticulin fibres at any time during the follow-up.[105] Consistently with patient data, alterations of TGF-β1, Hedgehog, and p53 signaling pathways were identified in the BM of GATA- 1low mice model.[106] Inhibition of TGF-β1 signaling in these mice by an inhibitor of the tyrosine kinase activity of TGF-β1 receptor type I, led to restoration of normal Mk development, reduced fibrosis, neoangiogenesis, and osteogenesis in the BM.[106] Based on these consistent observations, TGF-β inhibition has become a potential therapeutic strategy to decrease BM fibrosis in MPNs and is also being investigated in several clinical and experimental scenarios.[107]
Cxcl-4 (Pf-4). Cxcl-4, (C-X-C motif) ligand 4 (CXCL4) (also known as platelet factor 4 [PF4]), is one of the most abundant protein in the α-granules of Mk/platelets (estimated micromolar concentration), together with CXCL7.[108] This 70 a.a., cationic, lysine-rich, 7.8-kDa chemokine, is mainly synthesized by Mks, and comprises 2%-3% of the releasate from agonist-activated platelets. Once secreted, CXCL4 avidly binds to glycosaminoglycans, but only a splice-variant of the human chemokine receptor CXCR3 (CXCR3B), which is not present in mice, and LDLR90 have been identified as high-affinity receptors.[109] In contrast, the specific receptor for CXCL4 has not yet been identified in mice. However, in some circumstances, CXCL4 can interact with other chemokines (e.g. CCL5) and thereby modulate their effects on target cells. A central role of platelet-derived CXCL4 was demonstrated in solid organs. In vivo, mice lacking CXCL4 are significantly protected from severe liver fibrosis, demonstrating the pro-fibrotic phenotype of this chemokine and that its effects in mice are indeed mediated by other receptors than CXCR3.[110] In addition, it was shown that CXCL4 is secreted not only by activated platelets, but also by plasmacytoid dendritic cells and fibroblasts in systemic sclerosis.[111] Thus, these studies further involve Mks/platelets to pro- inflammatory and pro-fibrotic programmes in fibrosis. Interestingly, more than 30 years ago, Burstein et al. linked CXCL4 to myelofibrosis, by suggesting that abnormal Mks stimulate the proliferation of fibrosis-driving fibroblasts though the release of CXCL4.[112] However, no correlation was seen between BM fibrosis and plasma levels or the platelet content of CXCL4 in the same study.[112] Recently, Schneider et al. using a mouse model with genetic fate tracing in vivo, provided evidence that Gli1+ cells are key players in the initiation and progression of BM fibrosis and that Mk-derived CXCL4 was necessary and sufficient to induce the migration of Gli1+ stromal cells and their myofibroblastic differentiation.[113] In these experiments, CXCL4 was shown to induced myofibroblast differentiation of Gli1+ cells comparable to induction with TGF-β, a known stimulus for differentiation of MSCs into myofibroblasts.[113]
Other cytokines. Oncostatin M (OSM), is a pleiotropic cytokine belonging to the interleukin-6 (IL-6) family.[114] Produced mainly by activated T cells and monocytes, OSM can elicit different biological effects, depending on the cell type. OSM acts through two types of receptors. The type I OSM receptor is composed of gp130 and the leukemia inhibitory factor (LIF) receptor β-subunit (LIFR), and the type II OSM receptor is composed of gp130 and the OSM-specific receptor β- subunit (OSMR).[115] OSM has emerged as an important cytokine in the control of hematopoiesis. Transplantation experiments with OSM-deficient mice have shown that OSM stimulates stromal cells as well as hematopoietic progenitors and is required for the proper generation and maintenance of microenvironment in the BM.[116,117] Noteworthy, BM Mks express substantial amounts of OSM,[118] and OSM has been reported to behave as a megakaryocytic maturation factor in vitro and to augment platelet production in vivo.[119] Within the context of myelofibrosis, JAK2 V617F mutation promotes expression of OSM in neoplastic myeloid cells and, consequently, OSM mRNA levels are increased in the BM of patients with MPNs compared to control patients.[120] Mechanistically, OSM secreted by JAK2V617F+ cells stimulated growth of fibroblasts and endothelial cells by sustaining the production of angiogenic and pro-fibrogenic cytokines.[120]
Aberrant packaging of α-granule-specific proteins is supposed to trigger myelofibrosis in patients with GPS. Using a Nbeal2^-/- murine model of GPS, Guerrero et al. demonstrated that BM Mks from these mice were enriched in a restricted set of chemokines transcripts, namely CCL3 and CCL4, which encode macrophage inflammatory protein (MIP) 1α and 1β, respectively, well-known pro- inflammatory chemokines increased in PMF.[46] A peculiar role for MIP 1α in sustaining osteoblasts proliferation in MPN mice model has been also proposed.[121]
Interleukin 8 (IL-8) is a member of the family of chemokines related by a CXC motif. It binds to CXC chemokine receptor 1 (CXCR1) and 2 (CXCR2).[122] It is produced by several cell types, including Mks[123] and exhibits many biological functions in inflammation, HSC proliferation and mobilization and neo-angiogenesis. Increased levels of IL-8 were found in serum[124] and plasma[68] of patients with PMF. Additionally, IL-8 and its receptors were reported to be involved in PMF-altered Mk growth.[124] Finally, rodents lack a direct homologue of IL-8, but the chemokines CXCL1/KC, CXCL2/MIP-2, and CXCL5-6/LIX are regarded as functional homologues of IL-8.[125]
Finally,  Mks were repeatedly identified as the main cellular source of an increasing list of cytokines, which show higher plasma levels in PMF patients, and that are individually involved in the promotion of myelofibrosis. This list further includes bone morphogenic proteins (e.g. BMP- 1[126], BMP-2, -4, and -6[127]), Lipocalin-2 (LCN-2),[128] Fibroblasts Growth Factor (FGF),[129] Vascular Endothelial Growth Factor (VEGF),[130] Secreted Phospho Protein-1 (SPP1)[131] and Thrombospondin- 1 (TSP-1).[132,133]

Megakaryocyte expression of extracellular matrices and cross-linking enzymes in bone marrow fibrosis

Direct Megakaryocyte-cell interactions in the context of bone marrow fibrosis

Is the pro-fibrotic role of Megakaryocytes/ platelets restricted to the bone marrow? 

New discoveries in the field of thrombopoiesis and platelet roles have revealed unprecedented features of the Mk/Platelet lineage that open new avenues in the study of these cells, particularly in diseased conditions. Bioactive mediators, stored in platelets, have been implicated in fibrotic conditions that target solid organs, rather than BM (Table 2).

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Table 2. Megakaryocytes/Platelets contribution to organ fibrosis.

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A large amount of experimental evidence implies that platelets participate in the liver fibrotic process mainly by releasing pro- fibrotic mediators. Using mice carrying a Mk/platelet-specific targeted conditional deletion of the TGF-β1 gene (PF4^CreTgfβ1^f/f), Ghafoory et al. demonstrated that platelet TGF-β1 deficiency decreases liver fibrosis in a mouse model of carbon tetrachloride (CCl₄)-induced liver injury.[156] However, there is also evidence that platelets under certain circumstances may have a protective role against liver fibrosis. To this regard, thrombocytopenic mice, with selective disruption of the anti-apoptotic gene Bcl-xL, were shown to be more prone to liver fibrosis by bile duct ligation compared to their wild type counterparts.[157] The authors, suggested that the anti-fibrotic Hepatocyte Growth Factor (HGF) released from activated platelets in liver, attenuated the expression of collagen in hepatic stellate cells, the key cell type in liver fibrosis.[157] Additionally, Mk-specific disruption of the TGF-β1 gene resulted in mice protection from cardiac hypertrophy, fibrosis, and systolic dysfunction in response to transverse aortic constriction, suggesting that platelet profibrotic behavior is not solely restricted to the liver.[158] Similarly, evidence has been accumulated implicating platelets in the pathogenesis of interstitial lung fibrosis in several animal models. Piguet et al. found that trapping of platelets in contact with the alveolar endothelium of the lungs after bleomycin injection was increased and correlated with the deposition of collagen.[159] The authors suggested that this could represent not only a simple correlation but also a potential pathological mechanism that links platelets and pulmonary fibrosis. Interestingly, in a recent study platelets were shown to promote acute lung injury through the massive release of the Wnt/β-catenin inhibitor Dickkopf-1 (Dkk-1) from their α-granules, leading to increased expression of vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) on the surface of alveolar epithelial cells (AECs) and abnormal macrophage/neutrophils interaction with AECs.[160] In addition to the direct involvement of platelets in fibrosis of solid organs, the potential contribution of Mks to organ fibrosis still needs to be uncovered. It is becoming increasingly clear that Mks transfer unique genetic codes to platelets, and that environmental changes can alter transcriptional, translational, and post-translational processes in Mks, affecting the genetic code of platelets in circulation.[161] Change in Mk-platelet transcriptional axis is a dynamic process, especially in disease situations, and can rapidly affect the platelet repertoire of messenger RNAs (mRNAs), microRNAs (miRNAs), and proteins that contribute to their primary and alternative functions. Freishtat et al., revealed for the first time, that a de novo transcriptome is imparted to platelets by BM residing Mks during sepsis.[162] Septic Mks produce platelets with acutely altered mRNA profiles, and these platelets mediate lymphotoxicity via the potent cytotoxic serine protease, granzyme B.[162] Similarly, in the context of cancer, Mks of tumor-bearing mice endocytose circulating thrombospondin-1 (TSP-1) and increase its synthesis to produce platelets with elevated levels of TSP-1, one of the most potent angiogenesis inhibitors. These TSP-1-enriched platelets were shown to adhere to tumors and to act as potent inhibitors of angiogenesis and cancer growth.[163] Thus, similar changes may occur in fibrotic conditions, but this has not been demonstrated yet.

Conclusions

In this review, we summarized the involvement of the Mk lineage in the development of BM fibrosis. We now know that, in addition to genetic triggers, BM fibrosis is sustained by the intramedullary release of cytokines that are responsible for the abnormal activation of stromal cells, resulting in extensive deposits of reticulin and collagens. Mks are supposed to constitute the main source of these reactive cytokines. Abnormal Mk differentiation, apoptosis and emperipolesis were all proposed as major mechanisms for the enhanced release of cytokines with a fibrogenic potential. Unfortunately, mechanisms underlying Mk secretion, their relationships with other BM lineages and their functional activities in physiological conditions as well as during myelofibrosis progression, are not well understood to date. The first attempt to directly target the Mk lineage was shown, recently, to revert the disease in both Jak2V617F and MPLW515L mice models. Using a small molecule, the AURKA inhibitor MLN8237, that induce Mk polyploidization, differentiation, and subsequent apoptosis, the Crispino’s group demonstrated that the pharmacological induction of Mk maturation was beneficial in terms of reduced burden of immature Mks and amelioration of PMF features, including BM fibrosis.[164] Thus, developing drugs able to re-establish Mk normal function may represent a new strategy to treat the disease and, at the same time, to understand its pathogenic mechanisms (Figure  2).

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Figure 2. Schematic representation of potential novel mechanisms for MPN pathogenesis.

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Acknowledgments

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