CONSTITUTIVE PRODUCTION AND THROMBIN-INDUCED RELEASE OF VASCULAR ENDOTHELIAL GROWTH-FACTOR BY HUMAN MEGAKARYOCYTES AND PLATELETS

We have shown that coculture of bone marrow microvascular endothelial cells with hematopoietic progenitor tells results in proliferation and differentiation of megakaryocytes. In these long-term cultures, bone marrow microvascular endothelial cell monolayers maintain their cellul ar integrity in the absence of exogenous endothelial growth factors. B ecause this interaction may involve paracrine secretion of cytokines, we evaluated megakaryocytic cells for secretion of vascular endothelia l growth factor (VEGF). Megakaryocytes (CD41a(+)) were generated by ex vivo expansion of hematopoietic progenitor cells with kit ligand and thrombopoietin for 10 days and further purified with immunomagnetic mi crobeads. Using reverse transcription-PCR, we showed that megakaryocyt ic cell lines (Dami, HEL) and purified megakaryocytes expressed mRNA o f the three VEGF isoforms (121, 165, and 189 amino acids). Large quant ities of VEGF (>1 ng/10(6) cells/3 days) were detected in the supernat ant of Dami cells, ex vivo-generated megakaryocytes, and CD41a(+) cell s isolated from bone marrow. The constitutive secretion of VEGF by CD4 1a(+) cells was stimulated by growth factors of the megakaryocytic lin eage (interleukin 3, thrombopoietin), Western blotting of heparin-Seph arose-enriched supernatant mainly detected the isoform VEGF(165). In a ddition, immunohistochemistry showed intracytoplasmic VEGF in polyploi d megakaryocytes. Thrombin stimulation of megakaryocytes and platelets resulted in rapid release of VEGF within 30 min. We conclude that hum an megakaryocytes produce and secrete VEGF in an inducible manner. Wit hin the bone marrow microenvironment, VEGF secreted by megakaryocytes may contribute to the proliferation of endothelial cells. VEGF deliver ed to sites of vascular injury by activated platelets may initiate ang iogenesis.