Lysyl oxidase (LOX), a matrix cross-linking protein, is known to be selectively expressed and to enhance a fibrotic phenotype. A recent study of ours showed that LOX oxidizes the PDGF receptor-␤ (PDGFR-␤), leading to amplified downstream signaling. Here, we examined the expression and functions of LOX in megakaryocytes (MKs), the platelet precursors. Cells committed to the MK lineage undergo mitotic proliferation to yield diploid cells, followed by endomitosis and acquisition of polyploidy. Intriguingly, LOX expression is detected in diploid-tetraploid MKs, but scarce in polyploid MKs. PDGFR-BB is an inducer of mitotic proliferation in MKs. LOX inhibition with ␤-aminopropionitrile reduces PDGFR-BB binding to cells and downstream signaling, as well as its proliferative effect on the MK lineage. Inhibition of LOX activity has no influence on MK polyploidy. We next rationalized that, in a system with an abundance of low ploidy MKs, LOX could be highly expressed and with functional significance. Thus, we resorted to GATA-1 low mice, where there is an increase in low ploidy MKs, augmented levels of PDGF-BB, and an extensive matrix of fibers. MKs from these mice display high expression of LOX, compared with control mice. Importantly, treatment of GATA-1 low mice with ␤-aminopropionitrile significantly improves the bone marrow fibrotic phenotype, and MK number in the spleen. Thus, our in vitro and in vivo data support a novel role for LOX in regulating MK expansion by PDGF-BB and suggest LOX as a new potential therapeutic target for myelofibrosis. Lysyl oxidase (LOX)3 is a copper-dependent amine oxidase that catalyzes the oxidative deamination of lysine and hydroxylysine residues on collagen and elastin precursors. The resulting semialdehydes form covalent cross-linkages, thus stabilizing the extracellular matrix fiber deposits (1). LOX is synthesized as a 50-kDa glycosylated precursor (pro-LOX), which is then secreted and undergoes proteolytic cleavage by pro-collagen C-proteinases, including bone morphogenetic protein 1, to release a catalytically active 30-kDa enzyme (LOX) and an 18-kDa propeptide (2, 3). LOX has been associated with various pathologies, including cardiovascular diseases (4), neurodegenerative disorders (5, 6), and tumor progression and metastasis (7-9). An interesting insight into the regulation of cellular proliferation by LOX came from a recent study, showing that LOX can oxidize and activate cell surface proteins, including PDGFR-␤, in rat aortic smooth muscle cells (10, 11). Nevertheless, the role of this oxidase in regulating megakaryocyte (MK) expansion and/or ploidy had not been explored.Megakaryocytes (MKs), the platelet precursors, undergo proliferation followed by endomitosis and polyploidy, prior to fragmenting into platelets (12, 13). In certain pathologies, the proliferation and ploidy of this lineage are deregulated, highlighting the need to further elucidate mechanisms of control of these processes (14, 15). Thrombopoietin (TPO) is the primary growth factor that stimulates the prolif...

Summary. Background: Activation of platelets is a critical component of atherothrombosis and plays a central role in the progression of unstable cardiovascular syndromes. Adenosine, acting through adenosine receptors, increases intracellular cAMP levels and inhibits platelet aggregation. The A2a adenosine receptor has already been recognized as a mediator of adenosine-dependent effects on platelet aggregation, and here we present a new role for the A2b adenosine receptor (A2bAR) in this process. Methods and Results: As compared with platelets from wild-type controls, platelets derived from A2bAR knockout mice have significantly greater ADP receptor activation-induced aggregation. Although mouse megakaryocytes and platelets express low levels of the A2bAR transcript, this gene is highly upregulated following injury and systemic inflammation in vivo. Under these conditions, A2bAR-mediated inhibition of platelet aggregation significantly increases. Our studies also identify a novel mechanism by which the A2bAR could regulate platelet aggregation; namely, ablation of the A2bAR leads to upregulated expression of the P2Y1 ADP receptor, whereas A2bAR-mediated or direct elevation of cAMP has the opposite effect. Thus, the A2bAR regulates platelet function beyond mediating the immediate effect of adenosine on aggregation. Conclusions: Taken together, these investigations show for the first time that the platelet A2bAR is upregulated under stress in vivo, plays a significant role in regulating ADP receptor expression, and inhibits agonistinduced platelet aggregation.

Megakaryocytes are platelet precursor cells that undergo endomitosis. During this process, repeated rounds of DNA synthesis are characterized by lack of late anaphase and cytokinesis. Physiologically, the majority of the polyploid megakaryocytes in the bone marrow are cell cycle arrested. As previously reported, cyclin E is essential for megakaryocyte polyploidy; however, it has remained unclear whether up-regulated cyclin E is an inducer of polyploidy in vivo. We found that cyclin E is up-regulated upon stimulation of primary megakaryocytes by thrombopoietin. Transgenic mice in which elevated cyclin E expression is targeted to megakaryocytes display an increased ploidy profile. Examination of S phase markers, specifically proliferating cell nuclear antigen, cyclin A, and 5-bromo-2-deoxyuridine reveals that cyclin E promotes progression to S phase and cell cycling. Interestingly, analysis of Cdc6 and Mcm2 indicates that cyclin E mediates its effect by promoting the expression of components of the pre-replication complex. Furthermore, we show that up-regulated cyclin E results in the up-regulation of cyclin B1 levels, suggesting an additional mechanism of cyclin E-mediated ploidy increase. These findings define a key role for cyclin E in promoting megakaryocyte entry into S phase and hence, increase in the number of cell cycling cells and in augmenting polyploidization. Megakaryocytes (MKs)2 are bone marrow precursor cells responsible for the production of platelets, which are renewed on a daily basis (1). In a process termed megakaryopoiesis, multipotent hematopoietic stem cells commit toward becoming megakaryocyte progenitors. This is followed by differentiation of progenitors into mature MKs while concomitantly undergoing polyploidization (reviewed in Ref. being 16N (6 -7). During this process, MKs increase the production of proteins necessary for platelet biogenesis and function (8). Mature MKs form proplatelet extensions that fragment and give rise to platelets (9).The mechanism by which MKs become polyploid is still not well understood. Following a series of normal cell divisions, MKs enter a cell cycle with a brief G1 phase, followed by a typical S phase and a very short G2 phase (6). Next, MKs undergo endoreplication, which represents a mitotic cell cycle that is terminated at the late anaphase stage. Repeated rounds of endoreplication eventually give rise to a polyploid megakaryocyte. The regulatory mechanisms that control polyploidization have been partially explored, with a major focus on the regulation of mitotic phase and cytokinesis (10 -13). As to mitotic regulation, it became clear that it is different in MK cell lines and primary MKs, as the former show decreased levels of cyclin B in polyploid MKs (14 -15), while primary MKs display cyclin B during endomitotis (16 -17). Other studies have focused on the G1 phase of the MK cell cycle as a regulatory phase of polyploidy. The first study in this area showed that cyclin D3, which is highly expressed at early G1 phase, is up-regulated in MKs (18). C...

Background: ETV2 has been identified as an important player in embryonic hematopoiesis. However, the cell populations in which this transcription factor is expressed and operates during blood specification remain to be fully characterized. Here we address these issues using ES cells and a transgenic mouse line expressing green fluorescent protein (GFP) under the control of ETV2 regulatory elements, allowing us to observe the tight association between ETV2 expression and the initiation of hematopoiesis. Results: Both in differentiating ES cells and gastrulating embryos ETV2::GFP is mostly found co‐expressed with endothelial markers and defines a subset of cells with greatly enriched primitive erythroid potential. Upon culture ETV2::GFP cells rapidly up‐regulate CD41, down‐regulate endothelium cell surface markers and generate definitive hematopoietic progenitors. Altogether these characteristics represent the hallmark of hemogenic endothelium cells, a specialized endothelium originating from the hemangioblast and giving rise to hematopoietic cells. Importantly, ETV2 deficiency results in a complete absence of hemogenic endothelium in differentiating ES cells and gastrulating embryos. Conclusions: Altogether our data reveal that ETV2 marks hemogenic endothelium in gastrulating embryos and is absolutely required for the formation of this precursor at the onset of hematopoiesis. These results enhance our understanding of embryonic hematopoiesis and the factors driving hemogenic endothelium specification. Developmental Dynamics 241:1454–1464, 2012. © 2012 Wiley Periodicals, Inc.

SummaryIt is now well-established that hematopoietic stem cells (HSCs) and progenitor cells originate from a specialized subset of endothelium, termed hemogenic endothelium (HE), via an endothelial-to-hematopoietic transition. However, the molecular mechanisms determining which endothelial progenitors possess this hemogenic potential are currently unknown. Here, we investigated the changes in hemogenic potential in endothelial progenitors at the early stages of embryonic development. Using an ETV2::GFP reporter mouse to isolate emerging endothelial progenitors, we observed a dramatic decrease in hemogenic potential between embryonic day (E)7.5 and E8.5. At the molecular level, Runx1 is expressed at much lower levels in E8.5 intra-embryonic progenitors, while Bmi1 expression is increased. Remarkably, the ectopic expression of Runx1 in these progenitors fully restores their hemogenic potential, as does the suppression of BMI1 function. Altogether, our data demonstrate that hemogenic competency in recently specified endothelial progenitors is restrained through the active silencing of Runx1 expression.

IntroductionMegakaryocytes (MKs) are platelet precursors in the bone marrow (BM) that undergo a complex maturation process called megakaryopoiesis. A hallmark of MK development occurs after their commitment to the MK lineage, in which MKs go through iterative rounds of endomitosis to become large polyploid cells. These cells can contain DNA content of up to and greater than 128N, with 2N being normal diploid DNA content. 1 The primary stimulus for progression of megakaryopoiesis is known to stem from thrombopoietin (TPO) signaling through the c-Mpl receptor 2 ; however, despite this being the focus of many studies, much of TPO signaling in MKs remains unclear.Recently, investigations of reactive oxygen species (ROS), their generation, and downstream signaling effects have shown that low physiologic levels of ROS play a role in promoting cell proliferation in many cells types, such as fibroblast, prostate, macrophage, endothelial, and vascular smooth muscle cells (VSMCs). 3-7 ROSproducing enzymes, such as a member of the NADPH oxidase (Nox) family, Nox1, have been described to be activated by mitogenic stimuli 8 and to produce ROS that have been shown to induce and maintain G 1 phase cyclin D1 expression in mouse lung epithelial cells. 9 Similarly, Havens et al 10 showed that ROSs increase in a cell cycle-dependent manner and oscillate with every cell division in human T98G glioblastoma cells, human leukemic T cells, and NIH 3T3 cells. Although, these investigators did not identify the source of ROS in these cells, they did show that on treatment with various antioxidants, cells undergo transient arrest and fail to make a timely G 1 -S phase progression. [11][12][13] In addition, Havens et al 10 described redox control over the activity of the anaphase promoting complex (APC) in association with Cdh1, mediating cyclin A degradation, a regulator of the G 1 /S transition. In the MK endomitotic cell cycle, cyclin D3 has been described as the predominant D-type cyclin that is up-regulated by TPO stimulation and induces high ploidy levels in cyclin D3 transgenic mice. 14-16 Ablation of cyclin D3 in cultured MKs results in a notable decrease in MK ploidy level, 16 although in vivo studies show a less dramatic effect on ploidy possibly because of compensation by other D cyclins. 17 Cyclin E is another G 1 phase cell-cycle regulator that has been suggested to play a role in MK ploidy promotion, notably inducing nonendomitotic megakaryoblastic K562 cells to undergo re-replication cycles. 18 The importance of cyclin E in promotion of ploidy is also supported by evidence from the cyclin E1 Ϫ/Ϫ E2 Ϫ/Ϫ double knockout (KO) mice produced with the use of the tetraploid complementation rescue method. 19 Interestingly, these mice developed normally with the exception of ploidy promotion in trophoblasts and MKs, showing a marked reduction in polyploid MKs even when cultured with TPO.To date, investigation of ROS in MKs has been limited to studies that used human megakaryocytic leukemic cell lines, such as MO7e and B1647 cells. 11,...

• Mice overexpressing LOX in platelets have more severe thrombosis than normal animals.• LOX expression influences platelet adhesion to collagen. ) were generated. Pf4-Lox tg/tg mice had a normal number of platelets; however, time to vessel occlusion after endothelial injury was significantly shorter in Pf4-Lox tg/tg mice, indicating a higher propensity for thrombus formation in vivo.Exploring underlying mechanisms, we found that Pf4-Lox tg/tg platelets adhere better to collagen and have greater aggregation response to lower doses of collagen compared with controls. Platelet activation in response to the ligand for collagen receptor glycoprotein VI (cross-linked collagen-related peptide) was unaffected. However, the higher affinity of Pf4-Lox tg/tg platelets to the collagen sequence GFOGER implies that the collagen receptor integrin a 2 b 1 is affected by LOX. Taken together, our findings demonstrate that LOX enhances platelet activation and thrombosis. (Blood. 2016;127(11):1493-1501

Reactive oxygen species (ROS), generated as a result of various reactions, control an array of cellular processes. The role of ROS during megakaryocyte (MK) development has been a subject of interest and research. The bone marrow niche is the major site of MK differentiation and maturation. In this environment, a gradient of oxygen tension, from normoxia to hypoxia results in different levels of ROS, impacting cellular physiology. This article provides an overview of major sources of ROS, their implication in different signaling pathways, and their effect on cellular physiology, with a focus on megakaryopoiesis. The importance of ROS-generating oxidases in MK biology and pathology, including myelofibrosis, is also described.