Megakaryocytes produce vascular endothelial growth factor (VEGF): Increased release after stimulation and possible role in migration and platelet shedding
Endothelial cells play an important role in the growth and differentiation of megakaryocytes, and subsequent platelet release. Since interaction between endothelial uid nttgakaryocytic cells might involve paracrine secretion of cytokines, we analyzed expression of vascular endotbcJial growth factor (VEGF) in human megakaryocytic cell lines and megakaiyocytes generated by ex vivo-expanskm of honatopoietic progenitor cells with kit ligand (KL) and thrombopoietin (TPO) for 14 days. CD41a+ cells were fluther isolated with immunomagnetic microspheres to a purity >95%. By RT-PCR, the megakaryocytic cell lines Dami and HEL, as well as the purified megakaryocytes Iransacribed mRNA of the three alternatively spliced isofonns of VEGF (121,165, and 189 aminoaddc). Expression of VEGF mRNA in isolated megakaryocytei was not related to contaminating mydoid cells, since setniquantitative RT-PCR of total RNA from the CD4 la-negative cell fraction nulled in a weaker signal compared to CD41+ celli. Furthermore, conditioned médium from isolated megakaryocytes contained the greatest amount of VEGF (10.2 ng per 106 edit, incubated in 1 ml medium for 3 days, measured by ELISA assay) compared to CD4la-negative cells (<0,1 ng / ID6 cells) and Dami cells (4.1 ng / 106 cells). By Western Hotting of conditioned medium, the three alternatively spliced VEGF isofonns could also be detected at the protein level. Imrnunhistochemistry of Dami celli and megakaryocytes showed imracytoplasmic VEGF expression. Interestingly, the intensity of the staining was related to the cell size suggesting that large, polyploid megakaryocytes store VEGF protein. Indeed, stimulation of Kl/TPO expanded cells with thrombin resulted in a release of 2.2 ng VEGF /106 cells within 30 min compared to 0.5 ng / 106 cells without stimulation. In conclusion, megakaiyocytes produce VEGF and release the protein in a stimulation-dependent manner. Since VEGF, originally described as vascular permeability factor, leads to increased microvascular permeability, it may also play a role in migration of megakaryocytes and platelet release.