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Keywords:

  • chronic myeloid leukaemia;
  • polycythaemia vera;
  • Cox-2;
  • Tie-2;
  • glycodelin

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Cox-2 expression
  6. Glycodelin expression
  7. Tie-2 expression
  8. Discussion
  9. Acknowledgments
  10. References

Summary.  When evaluating bone marrow sections for markers of neo-angiogenesis, we found that megakaryocytes stained markedly positive for cyclooxygenase-2 (Cox-2), Tie-2 and glycodelin. This apparently novel finding was further evaluated for disease-specific variations. Bone marrow sections from two patient groups, known to be characterized by clonal megakaryocytopoiesis, viz. chronic myeloid leukaemia and polycythaemia vera, stained, however, similarly to healthy marrows for these markers. The biochemical background and clinical significance of Cox-2, Tie-2 and glycodelin remains to be elucidated.

Accelerated angiogenesis has been noted in the chronic myeloproliferative disorders, observed by an increased microvascular density (MVD) and staining for vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). These features are particularly evident in chronic myeloid leukaemia (CML) and myelofibrosis, whereas polycythaemia vera (PV) marrows may not display significantly increased vascularity, but show abnormal vascular architecture (Lundberg et al, 2000).

Recently, when evaluating a variety of new markers of neo-angiogenesis in bone marrows from patients diagnosed with chronic myeloproliferative disorders, we made the unexpected finding that megakaryocytes stained for three such markers. These were: Cyclooxygenase (Cox)-2, the inducible form of Cox that converts arachidonic acid to prostaglandins; Tie-2, the common surface receptor for angiopoietins 1 and 2 (Davis et al, 1996); and glycodelin, a 28-kDa glycoprotein, synthesized by endometrial tissue and decidua during pregnancy (Kamarainen et al, 1998). As both PV and CML are characterized by dysmegakaryocytopoesis, we extended the study to evaluate whether Cox-2, Tie-2 and glycodelin are specifically over- or underexpressed in either PV or CML.

Patients.  Bone marrow biopsies from seven patients with CML, seven patients with PV and from five healthy subjects were obtained for routine diagnostic procedures and retrieved as previously described (Lundberg et al, 2000). The control group consisted of patients who had been investigated because of diagnosis of either anaemia or leukocytosis, but who had a normal bone marrow according to routine examination by a specialized haemopathologist. The study was approved by the Huddinge University Hospital Ethics Committee.

Tissue preparation and immunohistochemistry.  Immunohistochemistry was performed on paraffin-embedded bone marrow. In short, sections were de-paraffinized in xylene, rehydrated by serial incubations with decreasing concentrations of ethanol and then heated in a microwave oven for 5 min.

The primary antibodies used here were: a monoclonal mouse anti-human Cox-2 antibody (Transduction Laboratories, CA, USA), a polyclonal rabbit anti-human antibody to Tie-2 (R & D Systems, Abingdon, UK) and a polyclonal chicken anti-human antibody to glykodelin (a kind gift from Dr S. Ramachandran, USA). The specificity of antibodies was confirmed by experiments performed by the supplier. Negative controls consisted of samples stained only with the secondary antibody or stained with an irrelevant idiotypic antibody. Antigen detection was performed using the StraViGen supersensitive kit (BioGenix, San Ramon, CA, USA), according to the instructions by the supplier. Diaminobenzidine (DAB) (BioGenix) was added as the substrate. Sections were counter-stained with haematoxylin. Positive immunostaining was visualized as a brown colour and results were obtained with a light microscope.

Cox-2 expression

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Cox-2 expression
  6. Glycodelin expression
  7. Tie-2 expression
  8. Discussion
  9. Acknowledgments
  10. References

In all samples, megakaryocytes stained markedly for Cox-2. The staining was evenly distributed throughout the cytoplasm. There was no difference in the staining intensity observed between patients and control subjects (Fig 1). As expected, megakaryocytes numbers were increased in PV and CML samples when compared with control subjects.

image

Figure 1. Megakaryocytes staining positive for Cox-2 (1a–c), glykodelin (2a–c) and tie-2 (3a–c) in bone marrow sections from a healthy subject (a), a patient with CML (b) and a patient with PV (c) (original magnification ×400).

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Glycodelin expression

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Cox-2 expression
  6. Glycodelin expression
  7. Tie-2 expression
  8. Discussion
  9. Acknowledgments
  10. References

Megakaryocytes stained positive for glycodelin in all samples (Fig 1). Staining was moderate to intense throughout the cytoplasm. There was no difference in the staining intensity between patients and control subjects.

Tie-2 expression

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Cox-2 expression
  6. Glycodelin expression
  7. Tie-2 expression
  8. Discussion
  9. Acknowledgments
  10. References

In all samples, the cytoplasm of megakaryocytes stained positive for Tie-2, but the staining intensity varied between individual cells, i.e. in some samples both strongly positive and totally negative megakaryocytes were found (Fig 1). This variability was not related to disease, as it was observed at similar rates in both patients and control sections.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Cox-2 expression
  6. Glycodelin expression
  7. Tie-2 expression
  8. Discussion
  9. Acknowledgments
  10. References

The presence of Cox-2 in megakaryocytes had not previously been reported. However, during the preparation of this manuscript it was reported that Cox-2, and the constitutively expressed Cox-1, are expressed in normal megakaryocytes (Rocca et al, 2002). We extend this observation to the two major myeloproliferative disorders, CML and PV, in which a similar level of Cox-2 expression was observed.

The expression of Tie-2 in megakaryocytes had not previously been observed. Tie-2 is either expressed as a cell membrane receptor on, for instance, endothelial cells, or as a soluble, circulating receptor (Reusch et al, 2001). It is unknown if the cytoplasmic distribution of Tie-2 observed in the present study represented newly formed Tie-2 molecules in the megakaryocytes or an uptake of soluble Tie-2. Further studies are required to determine the origin of the Tie-2.

Glycodelin has previously been shown to be expressed in a megakaryocytic cell line (Morrow et al, 1994). Here, we report of its presence in human bone marrow cells and of the observation of a similar expression intensity of the antigen between healthy subjects and patients with myeloproliferative disorders.

The present study demonstrates that Tie-2, Cox-2 and glycodelin are all expressed by megakaryocytes. Previously, the expression of these molecules has also been demonstrated in certain endothelial cells (Sato et al, 1993; Masferrer et al, 2000; Song et al, 2001). CML and PV are characterized by pathological angiogenesis, but may also display abnormal megakaryocytosis. We postulated that Tie-2, Cox-2 and glycodelin are abnormally regulated in megakaryocytes from patients with myeloproliferative disorders. However, using immunohistochemistry, we found no evidence to support this hypothesis.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Cox-2 expression
  6. Glycodelin expression
  7. Tie-2 expression
  8. Discussion
  9. Acknowledgments
  10. References

This work was supported by grants from The Swedish Medical Research Council (19X-05991, 71XS-13135), The Stockholm Cancer Society, King Gustav V:s Jubilee Fund, and The Swedish Association against Rheumatism, Karolinska Institutet, Huddinge University Hospital and Stockholm Söder Hospital.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Cox-2 expression
  6. Glycodelin expression
  7. Tie-2 expression
  8. Discussion
  9. Acknowledgments
  10. References
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