Catherine Strassel

Abnormal megakaryocyte morphology and proplatelet formation in mice with megakaryocyte-restricted MYH9 inactivation

Mutations in the MYH9 gene encoding nonmuscle myosin IIA lead to macrothrombocytopenia as observed in MYH9-related disorders. We used mice with megakaryocyte-restricted MYH9 inactivation to explore the role of myosin in thrombopoiesis. In situ, bone marrow MYH9Delta megakaryocytes were irregularly shaped, appearing leaky with poorly defined limits. The demarcation membranes were abnormally organized and poorly developed, pointing to an insufficient reservoir for the future formation of platelets. The cytoskeletal-rich peripheral zone was lacking due to the absence of the myosin filament network that normally surrounds the granular zone in wild-type cells. In vitro studies of cultured cells showed that MYH9Delta megakaryocytes were unable to form stress fibers upon adhesion to collagen, suggesting that the leaky shape results from defects in internal tension and anchorage to the extracellular environment. Surprisingly, the proportion of cells extending proplatelets was increased in MYH9Delta megakaryocytes and the proplatelet buds were larger. Overall, this study provides evidence for a role of myosin in different steps of megakaryocyte development through its participation in the maintenance of cell shape, formation and organization of the demarcation membranes and the peripheral zone, anchorage to the extracellular matrix, and proplatelet formation.

Intrinsic impaired proplatelet formation and microtubule coil assembly of megakaryocytes in a mouse model of Bernard-Soulier syndrome

Bernard-Soulier syndrome is caused by a deficiency of the platelet surface glycoprotein Ib-IX-V complex resulting in a severe platelet function defect. However this is compounded by a reduction in platelet numbers and the platelets in the circulation are abnormally large. As for many other platelet disorders, recent evidence and findings of this study suggest that the defective morphology has its origins in megakaryocyte function. See related perspective article on page 756. Background Giant platelets and thrombocytopenia are invariable defects in the Bernard-Soulier syndrome caused by deficiency of the GPIb-V-IX complex, a receptor for von Willebrand factor supporting platelet adhesion to the damaged arterial wall. Various properties of this receptor may be considered potential determinants of the macrothrombocytopenia. Design and Methods To explore the underlying mechanisms of the disease, megakaryopoiesis was studied in a mouse model deficient in GPIbβ. Megakaryocytes were initially characterized in situ in the bone marrow of adult mice, after which their capacity to differentiate into proplatelet-bearing cells was evaluated in cultured fetal liver cells. Results The number of megakaryocyte progenitors, their differentiation and progressive maturation into distinct classes and their level of endoreplication were normal in GPIbβ−/− bone marrow. However, the more mature cells exhibited ultrastructural anomalies with a thicker peripheral zone and a less well developed demarcation membrane system. GPIbβ−/− megakaryocytes could be differentiated in culture from Lin− fetal liver cells in normal amounts but the proportion of cells able to extend proplatelets was decreased by 41%. Moreover, the GPIbβ−/− cells extending proplatelets displayed an abnormal morphology characterized by fewer pseudopodial extensions with thicker shaft sections and an increased diameter of the terminal coiled elements. GPIbβ−/− released platelets were larger but retained a typical discoid shape. Proplatelet formation was similarly affected in bone marrow explants from adult mice examined by videomicroscopy. The marginal microtubular ring contained twice as many tubulin fibers in GPIbβ−/− proplatelet buds in cultured and circulating platelets. Conclusions Altogether, these findings point to a role of the GPIb-V-IX complex intrinsic to megakaryocytes at the stage of proplatelet formation and suggest a functional link with the underlying microtubular cytoskeleton in platelet biogenesis.

Importance of environmental stiffness for megakaryocyte differentiation and proplatelet formation

Megakaryocyte (MK) differentiation occurs within the bone marrow (BM), a complex 3-dimensional (3D) environment of low stiffness exerting local external constraints. To evaluate the influence of the 3D mechanical constraints that MKs may encounter in vivo, we differentiated mouse BM progenitors in methylcellulose (MC) hydrogels tuned to mimic BM stiffness. We found that MKs grown in a medium of 30- to 60-Pa stiffness more closely resembled those in the BM in terms of demarcation membrane system (DMS) morphological aspect and exhibited higher ploidy levels, as compared with MKs in liquid culture. Following resuspension in a liquid medium, MC-grown MKs displayed twice as much proplatelet formation as cells grown in liquid culture. Thus, the MC gel, by mimicking external constraints, appeared to positively influence MK differentiation. To determine whether MKs adapt to extracellular stiffness through mechanotransduction involving actomyosin-based modulation of the intracellular tension, myosin-deficient (Myh9-/-) progenitors were grown in MC gels. Absence of myosin resulted in abnormal cell deformation and strongly decreased proplatelet formation, similarly to features observed for Myh9-/- MKs differentiated in situ but not in vitro. Moreover, megakaryoblastic leukemia 1 (MKL1), a well-known actor in mechanotransduction, was found to be preferentially relocated within the nucleus of MC-differentiated MKs, whereas its inhibition prevented MC-mediated increased proplatelet formation. Altogether, these data show that a 3D medium mimicking BM stiffness contributes, through the myosin IIA and MKL1 pathways, to a more favorable in vitro environment for MK differentiation, which ultimately translates into increased proplatelet production.

Aryl hydrocarbon receptor–dependent enrichment of a megakaryocytic precursor with a high potential to produce proplatelets

The mechanisms regulating megakaryopoiesis and platelet production (thrombopoiesis) are still incompletely understood. Identification of a progenitor with enhanced thrombopoietic capacity would be useful to decipher these mechanisms and to improve our capacity to produce platelets in vitro. Differentiation of peripheral blood CD34(+) cells in the presence of bone marrow-human mesenchymal stromal cells (MSCs) enhanced the production of proplatelet-bearing megakaryocytes (MKs) and platelet-like elements. This was accompanied by enrichment in a MK precursor population exhibiting an intermediate level of CD41 positivity while maintaining its expression of CD34. Following sorting and subculture with MSCs, this CD34(+)CD41(low) population was able to efficiently generate proplatelet-bearing MKs and platelet-like particles. Similarly, StemRegenin 1 (SR1), an antagonist of the aryl hydrocarbon receptor (AhR) transcription factor known to maintain CD34 expression of progenitor cells, led to an enriched CD34(+)CD41(low) fraction and to an increased capacity to generate proplatelet-producing MKs and platelet-like elements ultrastructurally and functionally similar to circulating platelets. The effect of MSCs, like that of SR1, appeared to be mediated by an AhR-dependent mechanism because both culture conditions resulted in repression of its downstream effector CYP1B1. This newly described isolation of a precursor exhibiting strong MK potential could be exploited to study normal and abnormal thrombopoiesis and for in vitro platelet production.

Broader expression of the mouse platelet factor 4‐cre transgene beyond the megakaryocyte lineage

Transgenic mice expressing cre recombinase under the control of the platelet factor 4 (Pf4) promoter, in the context of a 100‐kb bacterial artificial chromosome, have become a valuable tool with which to study genetic modifications in the platelet lineage. However, the specificity of cre expression has recently been questioned, and the time of its onset during megakaryopoiesis remains unknown.

Characterization of megakaryocyte development in the native bone marrow environment.

Differentiation and maturation of megakaryocytes occur in close association with cellular and extracellular components in the bone marrow. Thus, direct examination of these processes in the native environment provides important information regarding the development of megakaryocytes. In this chapter, we present methods applied to mouse bone marrow to (1) examine the ultrastructure of megakaryocytes and their state of maturation in situ in fixed bone marrow sections and (2) study the dynamics of proplatelet formation by real-time observation of fresh bone marrow explants where megakaryocytes have matured in their natural physiological context. Combining these two approaches allows detailed investigation of in situ megakaryocyte differentiation, including proplatelet formation, which is the final maturation step before platelet release.

Studies of mice lacking the GPIb-V-IX complex question the role of this receptor in atherosclerosis

Atherosclerosis is a multifactorial disease resulting from a complex interplay of actors such as lipids, monocytes, vascular endothelial and smooth muscle cells, lymphocytes and platelets [1]. Blood platelets not only play a crucial role in hemostasis and thrombus formation but are also implicated in the early stages of atherogenesis [2]. This role has been known for a long time from the observation of decreased initiation of atherosclerosis in animals rendered thrombocytopenic by injection of antiplatelet antibodies [3]. Platelets participation has been linked to their capacity to mediate the delivery of proinflammatory factors to monocytes/leukocytes and the vessel wall, and accordingly, administration of activated platelets to ApoEdeficient mice (ApoE) has been shown to exacerbate atherosclerosis [4]. Along the same lines, genetic ablation of platelet secreted factors [2,5] (PF-4, CD40-L, RANTES) [6–8], or prevention of their interaction with leukocytes via exposed P-selectin [9], has resulted in a lower tendency to develop atherosclerotic plaques. In addition to their activation capacity, adhesive functions have also been implicated in platelet proatherogenic properties. Among them, an important role has recently been attributed to the GPIb-V-IX axis from the profoundly decreased development of atherosclerotic lesions in hyperlipidemic ApoEmice injected with an anti-GPIba antibody [10]. Although the precise implication of GPIb is complicated by the important thrombocytopenia induced by these antibodies injected as IgGs [11], administration of non-thrombocytopenic Fab fragments reduced monocyte interaction with vascular endothelium in vivo [10,11]. The capacity of GPIba to bind von Willebrand factor (VWF) and P-selectin on activated subendothelium and to bind aMb2-integrin on monocytes/leukocytes provides a biological framework supporting its role in monocyte recruitment in the early stages of plaque development [12,13]. Mice with deficiencies of the GPIb-V-IX complex after genetic ablation of the GPIba or GPIbb subunit are now available [14,15], giving the opportunity to analyze further the role of this receptor in atherogenesis. Atherosclerotic plaque development was examined in GPIbb mice (backcrossed seven times to C57BL/6J) crossed to ApoE mice (Charles River, L Arbresle, France) to generate GPIbb/ApoE doubleknockout mice (GPIbb/ApoE), which reproduced the platelet phenotype reported previously in the mixed GPIbb strain [14]. These mice did not express the GPIb-V-IX complex on the surface and also exhibited decreased platelet counts with levels representing 15% of those in ApoE controls [1423.10 ± 85.10 platelets lL in ApoE (n = 8) vs. 205.10 ± 17.10 platelets lL in GPIbb/ApoE mice (n = 9)]. Although thrombocytopenia represents a potential limitation, as it could inhibit plaque formation independently of GPIb, this strain nevertheless represents a very useful and complementary model to appreciate the role of this receptor in atherogenesis. These mice were maintained on regular mouse chow containing 3% fat for 17 or 30 weeks before collection of the whole aortas and hearts for analysis of the size and composition of atherosclerotic lesions [16]. ApoE and GPIbb/ApoE mice exhibited similar body weights, and were hyperlipidemic but GPIbb/ApoE mice exhibited lower levels of cholesterol and triglycerides as compared with ApoE(Table S1). Extension of the atherosclerotic lesions was first analysed by en face staining with Oil Red O of a large segment of the descending aorta, from the subclavian branch to the iliac bifurcation. In this low fat chow diet, a limited area was stained with Oil Red O at 17 weeks, which was mostly observed at bifurcations and did not differ between the two strains (data not shown). At 30 weeks, an increased fraction of the surface was occupied by the lesions, which ranged in appearance from simply fatty streaks to complex fibrous plaques. Lesion area did not differ significantly as a whole between the two strains [18.3 ± 1.9% in ApoE (n = 10) vs. 17.8 ± 1.4% in GPIbb/ApoEmice (n = 10);P = 0.83] (Fig. 1A). Comparison restricted to the more lipid-rich aortic arches also did not reveal differences in plaque area between the two strains (data not shown). Atherosclerosis was analyzed in a second territory, the aortic sinus, which is particularly prone to intimal lesion, and allows both quantitative and qualitative assessment of plaque development. Analysis was performed on transversal sections guided by the positioning cue provided by the cups of the valves. The extent of the lesion was quantified on van Gieson stained cross-sections by measuring the entire surface of the Corresspondence: Catherine Strassel, UMR_S949 INSERMUniversite de Strasbourg, EFS-Alsace, Strasbourg, France. Tel.: +33 388212521. E-mail: catherine.strassel@efs-alsace.fr

Hirudin and heparin enable efficient megakaryocyte differentiation of mouse bone marrow progenitors

Hematopoietic progenitors from murine fetal liver efficiently differentiate in culture into proplatelet-producing megakaryocytes and have proved valuable to study platelet biogenesis. In contrast, megakaryocyte maturation is far less efficient in cultured bone marrow progenitors, which hampers studies in adult animals. It is shown here that addition of hirudin to media containing thrombopoietin and serum yielded a proportion of proplatelet-forming megakaryocytes similar to that in fetal liver cultures (approximately 50%) with well developed extensions and increased the release of platelet particles in the media. The effect of hirudin was maximal at 100 U/ml, and was more pronounced when it was added in the early stages of differentiation. Hirugen, which targets the thrombin anion binding exosite I, and argatroban, a selective active site blocker, also promoted proplatelet formation albeit less efficiently than hirudin. Heparin, an indirect thrombin blocker, and OTR1500, a stable heparin-like synthetic glycosaminoglycan generated proplatelets at levels comparable to hirudin. Heparin with low affinity for antithrombin was equally as effective as standard heparin, which indicates antithrombin independent effects. Use of hirudin and heparin compounds should lead to improved culture conditions and facilitate studies of platelet biogenesis in adult mice.

Cdc42-dependent F-actin dynamics drive structuration of the demarcation membrane system in megakaryocytes.

Essentials Information about the formation of the demarcation membrane system (DMS) is still lacking. We investigated the role of the cytoskeleton in DMS structuration in megakaryocytes. Cdc42/Pak‐dependent F‐actin remodeling regulates DMS organization for proper megakaryopoiesis. These data highlight the mandatory role of F‐actin in platelet biogenesis.

Dual role of IL-21 in megakaryopoiesis and platelet homeostasis

Gene profiling studies have indicated that in vitro differentiated human megakaryocytes express the receptor for IL-21 (IL-21R), an immunostimulatory cytokine associated with inflammatory disorders and currently under evaluation in cancer therapy. The aim of this study was to investigate whether IL-21 modulates megakaryopoiesis. We first checked the expression of IL-21 receptor on human bone marrow and in vitro differentiated megakaryocytes. We then investigated the effect of IL-21 on the in vitro differentiation of human blood CD34+ progenitors into megakaryocytes. Finally, we analyzed the consequences of hydrodynamic transfection-mediated transient expression of IL-21, on megakaryopoiesis and thrombopoiesis in mice. The IL-21Rα chain was expressed in human bone marrow megakaryocytes and was progressively induced during in vitro differentiation of human peripheral CD34+ progenitors, while the signal transducing γ chain was down-regulated. Consistently, the STAT3 phosphorylation induced by IL-21 diminished during the later stages of megakaryocytic differentiation. In vitro, IL-21 increased the number of colony-forming unit megakaryocytes generated from CD34+ cells and the number of megakaryocytes differentiated from CD34+ progenitors in a JAK3- and STAT3-dependent manner. Forced expression of IL-21 in mice increased the density of bi-potent megakaryocyte progenitors and bone marrow megakaryocytes, and the platelet generation, but increased platelet clearance with a consequent reduction in blood cell counts. Our work suggests that IL-21 regulates megakaryocyte development and platelet homeostasis. Thus, IL-21 may link immune responses to physiological or pathological platelet-dependent processes.