Nel R. Blom

BASAL TISSUE FACTOR EXPRESSION IN ENDOTHELIAL-CELL CULTURES IS CAUSED BY CONTAMINATING SMOOTH-MUSCLE CELLS - REDUCTION BY USING CHYMOTRYPSIN INSTEAD OF COLLAGENASE

A discrepancy exists between basal tissue factor (TF) expression found in endothelial cell cultures and the failure to detect TF in unpertubated endothelial cells in vivo. We demonstrated that basal TF expression in endothelial cell cultures originated from contaminating cells. These cells were ultrastructurally and flowcytometrically identified as smooth muscle cells. The cell cultures had been obtained from collagenase-treated human umbilical cord vessels. Histologic studies revealed that after collagenase treatment the basement membrane was digested and underlying structures were disrupted at some areas of the vein. We selected chymotrypsin as an alternative for the isolation of endothelial cells. Using chymotrypsin, the endothelial lining was selectively lost leaving the basement membrane undisturbed. Furthermore, use of chymotrypsin instead of collagenase minimized the level of basal TF activity. Taken together, we demonstrated that basal TF expression in endothelial cell cultures is caused by contaminating smooth muscle cells. This contamination can strongly be reduced using chymotrypsin instead of collagenase for isolation of endothelial cells.

Megakaryocytic dysfunction in myelodysplastic syndromes and idiopathic thrombocytopenic purpura is in part due to different forms of cell death

Platelet production requires compartmentalized caspase activation within megakaryocytes. This eventually results in platelet release in conjunction with apoptosis of the remaining megakaryocyte. Recent studies have indicated that in low-risk myelodysplastic syndromes (MDS) and idiopathic thrombocytopenic purpura (ITP), premature cell death of megakaryocytes may contribute to thrombocytopenia. Different cell death patterns have been identified in megakaryocytes in these disorders. Growing evidence suggests that, besides apoptosis, necrosis and autophagic cell death, may also be programmed. Therefore, programmed cell death (PCD) can be classified in apoptosis, a caspase-dependent process, apoptosis-like, autophagic and necrosis-like PCD, which are predominantly caspase-independent processes. In MDS, megakaryocytes show features of necrosis-like PCD, whereas ITP megakaryocytes demonstrate predominantly characteristics of apoptosis-like PCD (para-apoptosis). Triggers for these death pathways are largely unknown. In MDS, the interaction of Fas/Fas-ligand might be of importance, whereas in ITP antiplatelet autoantibodies recognizing common antigens on megakaryocytes and platelets might be involved. These findings illustrate that cellular death pathways in megakaryocytes are recruited in both physiological and pathological settings, and that different forms of cell death can occur in the same cell depending on the stimulus and the cellular context. Elucidation of the underlying mechanisms might lead to novel therapeutic interventions.

Mitochondrial disruption and limited apoptosis of erythroblasts are associated with high risk myelodysplasia. An ultrastructural analysis

AIMS To investigate the ultrastructural characteristics of erythroblasts in myelodysplasia (MDS) which might be of additional importance in understanding its pathogenesis. METHODS AND RESULTS 22 patients were classified according to FAB (French-American-British classification), IPSS (international prognostic score system), cytogenetic risk factors and transfusion dependency. Using electron microscopy, in 77% of the cases, nuclear abnormalities consisting of disrupted membranes and cystic/dilated perinuclear spaces were noted. In a limited number of patients (n=7), a low percentage of apoptosis in the erythroid lineage (mean 3.1+/-1.6%; median 3%: range 1-6) (normal controls: <0.5%) could be noted, primarily in mature erythroblasts and significantly associated with spongiform nuclear features. In all patients extensive cytoplasmic vacuolization and myelin figures in erythroblasts were demonstrated. In 55% of the cases, enlarged and abnormal mitochondria were observed, significantly associated with iron-accumulation. A significant inverse relation existed between the absence of apoptosis and more advanced, or high risk disease and cytogenetic risk factors. Mitochondrial abnormalities were significantly correlated with high risk disease as well with an increase in transfusion dependency. CONCLUSIONS These data indicate that in MDS apoptosis may play a role in early stages of disease. The overall prominent defects in mitochondria might be an additional defect that is involved in ineffective erythropoiesis.

Plant Sterols Cause Macrothrombocytopenia in a Mouse Model of Sitosterolemia

Mutations in either ABCG5 or ABCG8 cause sitosterolemia, an inborn error of metabolism characterized by high plasma plant sterol concentrations. Recently, macrothrombocytopenia was described in a number of sitosterolemia patients, linking hematological dysfunction to disturbed sterol metabolism. Here, we demonstrate that macrothrombocytopenia is an intrinsic feature of murine sitosterolemia. Abcg5-deficient (Abcg5-/-) mice showed a 68% reduction in platelet count, and platelets were enlarged compared with wild-type controls. Macrothrombocytopenia was not due to decreased numbers of megakaryocytes or their progenitors, but defective megakaryocyte development with deterioration of the demarcation membrane system was evident. Lethally irradiated wild-type mice transplanted with bone marrow from Abcg5-/- mice displayed normal platelets, whereas Abcg5-/- mice transplanted with wild-type bone marrow still showed macrothrombocytopenia. Treatment with the sterol absorption inhibitor ezetimibe rapidly reversed macrothrombocytopenia in Abcg5-/- mice concomitant with a strong decrease in plasma plant sterols. Thus, accumulation of plant sterols is responsible for development of macrothrombocytopenia in sitosterolemia, and blocking intestinal plant sterol absorption provides an effective means of treatment.

The in vitro effects of cytokines on expansion and migration of megakaryocyte progenitors

Increasing the number of megakaryocyte progenitors in stem cell transplants by ex vivo expansion culture may be an approach to accelerate platelet recovery in patients undergoing high‐dose chemotherapy. We evaluated the effect of three different cytokine combinations on expansion, with special emphasis on the type of colony formation and migration of megakaryocytic cells. The number of clonogenic megakaryocyte progenitors (colony‐forming units–megakaryocyte; CFU‐Mk) with high‐ (> 20 cells/colony) and low‐proliferative capacity (5–20 cells/colony) and the number of megakaryocytic (CD61+) cells were significantly increased by including interleukin 3 (IL‐3) or IL‐3 + IL‐6 + IL‐11 + Flt3‐ligand to cultures containing megakaryocyte growth and development factor (MGDF) plus stem cell factor (SCF). No difference in the maturation of megakaryocytes from all three cytokine combinations to platelets were observed, as demonstrated by electron microscopy. In chemotaxis experiments, the migration towards stromal cell‐derived factor 1 (SDF‐1) was shown to be reduced for CD61+ cells and megakaryocyte progenitors cultured in other cytokines besides MGDF + SCF. The reduced migration was related to a lower expression of CXCR4, the receptor for SDF‐1, on megakaryocytes from the proliferating cultures. These in vitro results demonstrate that expansion in IL‐3 and other cytokines besides MGDF + SCF significantly impair the capacity of megakaryocytic cells to migrate.

The reduced GM-CSF priming of ROS production in granulocytes from patients with myelodysplasia is associated with an impaired lipid raft formation

Patients with myelodysplasia (MDS) show an impaired reactive oxygen species (ROS) production in response to fMLP stimulation of GM‐CSF‐primed neutrophils. In this study, we investigated the involvement of lipid rafts in this process and showed that treatment of neutrophils with the lipid raft‐disrupting agent methyl‐β‐cyclodextrin abrogates fMLP‐induced ROS production and activation of ERK1/2 and protein kinase B/Akt, two signal transduction pathways involved in ROS production in unprimed and GM‐CSF‐primed neutrophils. We subsequently showed that there was a decreased presence of Lyn, gp91phox, and p22phox in lipid raft fractions from neutrophils of MDS. Furthermore, the plasma membrane expression of the lipid raft marker GM1, which increases upon stimulation of GM‐CSF‐primed cells with fMLP, was reduced significantly in MDS patients. By electron microscopy, we showed that the fMLP‐induced increase in GM1 expression in GM‐CSF‐primed cells was a result of de novo synthesis, which was less efficient in MDS neutrophils. Taken together, these data indicate an involvement of lipid rafts in activation of signal transduction pathways leading to ROS production and show that in MDS neutrophils, an impaired lipid raft formation in GM‐CSF‐primed cells results in an impaired ROS production.

Distinct roles of the mTOR components Rictor and Raptor in MO7e megakaryocytic cells

Objective:  During megakaryopoiesis, hematopoietic progenitor cells in the bone marrow proliferate and ultimately differentiate in mature megakaryocytes (MK). We and others have recently described a role for the mammalian target of Rapamycin (mTOR) in proliferation and differentiation of MK cells. Two non‐redundant complexes of mTOR have been described; mTORC1 containing rapamycin‐associated TOR protein (Raptor) and mTORC2 containing Rapamycin‐insensitive companion of mTOR (Rictor). The individual roles of these complexes in MK development have so far not been elucidated, and were investigated in this study.

Sinusoidal endothelial cells are damaged and display enhanced autophagy in myelodysplastic syndromes

Terminally differentiated bone marrow cells in low-risk myelodysplasia (MDS) undergo enhanced cell death leading to ineffective haematopoiesis. The cell death type may be cell-specific: studies in MDS erythroblasts have shown enhanced apoptosis but also autophagy, while megakaryocytes undergo a caspase-3 independent non-apoptotic death (Houwerzijl et al, 2009). A strong connection has been shown between the erythroid and endothelial lineage. Erythroid and endothelial cells might originate from a common precursor cell (Lancrin et al, 2009) and in MDS haematopoietic and circulating endothelial cells can carry similar chromosomal abnormalities (Della Porta et al, 2008). These findings raise the question whether endothelial cells in MDS might also be prone to cell death pathways and might have a disrupted architecture. To investigate this question in more detail, particularly in order to quantify vascularisation and study cell ultrastructure, immunohistochemistry and electron microscopy were performed on bone marrow samples of patients with refractory anaemia (RA, n = 3), refractory cytopenias with multilineage dysplasia (RCMD, n = 5), RA with ringed sideroblasts (RARS, n = 3), RCMD with ringed sideroblasts (RCMD-RS, n = 5), RA with excess blasts type 1 (RAEB-1, n = 2), acute myeloid leukaemia (AML, n = 9), and healthy controls (n = 4). Three AML patients had a proven prophase of MDS. According to the International Prognostic Scoring System (IPSS) patients with RA, RARS, RCMD, and RCMDRS (n = 16) were categorized as lower-risk, i.e. low risk (n = 6) and intermediate risk-1 (n = 10). The RAEB-1 patients were high-risk (n = 2). For patient characteristics see Table SI. CD34-immunostaining of MDS bone marrow biopsies demonstrated increased microvessel density (MVD; supporting materials and methods). In both RA/RCMD (n = 8) and RARS/RCMD-RS (n = 5) MVD was significantly increased compared to normal marrow [number of vessels: 15 1 4 4 (mean SD) and 12 1 7 3/power field respectively, versus 3 5 2 9 in healthy controls, P < 0 05]. MVD in high-risk MDS (n = 2) and AML (n = 6) was also significantly increased (18 7 6 6 and 13 1 4 8 respectively, P < 0 05) compared to normal, but not significantly different from low-risk MDS (see Fig 1). Vascular endothelial growth factor (VEGF) staining of endothelial cells was evaluated semiquantitively. Normal bone marrow endothelial cells demonstrated no to weak staining of VEGF, while the VEGF intensity of endothelial cells in RA/RCMD (n = 6), RARS/RCMD-RS (n = 6), and AML (n = 2) was significantly increased and varied between weak to strong indicating that, in line with the increased MVD, VEGF expression by MDS endothelial cells is significantly elevated (see Fig 1). Ultrastructural analysis of endothelial cells was performed on haematons of a subgroup of the lower-risk MDS (RA: n = 3, RCMD: n = 2, RARS: n = 3, RCMD-RS: n = 4) and AML patients (n = 5) and compared to normal bone marrow (n = 3). Haematons are compact particles containing haematopoietic progenitor cells residing within a stromal framework, including adipocytes, mesenchymal cells, macrophages and endothelial cells (Blazsek et al, 2000, and Fig S1). Haematons can be isolated from the spicules from the bone marrow aspirate. In haematons sinusoidal endothelial cells can be observed in an architecturally preserved form. In MDS patients, endothelial cells demonstrated irregular cell membranes with numerous small protrusions and frequent sprouting, compatible with neoangiogenesis (Table SII). In AML there was less sprouting and membrane outlines were smoother, however extensive networks of microvessels were observed. Abnormally shaped endothelial cells without pericytes and with degradation of the basal membrane were observed in both MDS and AML. Extensive cytoplasmic vacuolization was present in MDS endothelial cells, especially in RARS and RCMS-RS. The majority of these vacuoles had double membranes, a characteristic of autophagy. In addition, an increased number of large and dense secondary lysosomes were present in the damaged MDS endothelial cells (Fig 2 and Table SII). Mitochondria were mainly ultrastructurally normal. No features of apoptosis were found in MDS endothelial cells, and this was confirmed by a negative immunostaining for caspase-3 and caspase-8 (data not shown). LC3, a marker of autophagic membranes (see supplementary material and methods), could be demonstrated on membranes and inside the majority of the vacuoles in MDS endothelial cells (Fig 2). In AML, autophagy was also observed in endothelial cells but at a low level, which might be linked to the prophase of MDS in 33% of the AML patients. Thus, enhanced autophagy was not a general phenomenon related to an increase in MVD. It is likely that in both MDS and AML the increased MVD might change bone marrow blood flow, thereby triggering hypoxia leading to upregulation of