Möhle R, Green D, Moore M A, Nachman R L, Rafii S
Developmental Hematopoiesis Laboratory, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.
Proc Natl Acad Sci U S A. 1997 Jan 21;94(2):663-8. doi: 10.1073/pnas.94.2.663.
We have shown that coculture of bone marrow microvascular endothelial cells with hematopoietic progenitor cells results in proliferation and differentiation of megakaryocytes. In these long-term cultures, bone marrow microvascular endothelial cell monolayers maintain their cellular integrity in the absence of exogenous endothelial growth factors. Because this interaction may involve paracrine secretion of cytokines, we evaluated megakaryocytic cells for secretion of cytokines, we evaluated megakaryocytic cells for secretion of vascular endothelial 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 microbeads. Using reverse transcription-PCR, we showed that megakaryocytic cell lines (Dami, HEL) and purified megakaryocytes expressed mRNA of the three VEGF isoforms (121, 165, and 189 amino acids). Large quantities of VEGF (> 1 ng/10(6) cells/3 days) were detected in the supernatant of Dami cells, ex vivo-generated megakaryocytes, and CD41a+ cells isolated from bone marrow. The constitutive secretion of VEGF by CD41a+ cells was stimulated by growth factors of the megakaryocytic lineage (interleukin 3, thrombopoietin). Western blotting of heparin-Sepharose-enriched supernatant mainly detected the isoform VEGF165. In addition, immunohistochemistry showed intracytoplasmic VEGF in polyploid megakaryocytes. Thrombin stimulation of megakaryocytes and platelets resulted in rapid release of VEGF within 30 min. We conclude that human megakaryocytes produce and secrete VEGF in an inducible manner. Within the bone marrow microenvironment, VEGF secreted by megakaryocytes may contribute to the proliferation of endothelial cells. VEGF delivered to sites of vascular injury by activated platelets may initiate angiogenesis.
我们已经证明，骨髓微血管内皮细胞与造血祖细胞共培养可导致巨核细胞增殖和分化。在这些长期培养中，骨髓微血管内皮细胞单层在没有外源性内皮生长因子的情况下维持其细胞完整性。由于这种相互作用可能涉及细胞因子的旁分泌，我们评估了巨核细胞分泌细胞因子的情况，还评估了巨核细胞分泌血管内皮生长因子（VEGF）的情况。通过用kit配体和血小板生成素对造血祖细胞进行体外扩增10天来生成巨核细胞（CD41a+），并用免疫磁珠进一步纯化。使用逆转录-聚合酶链反应，我们表明巨核细胞系（Dami、HEL）和纯化的巨核细胞表达三种VEGF异构体（121、165和189个氨基酸）的mRNA。在Dami细胞、体外生成的巨核细胞以及从骨髓中分离的CD41a+细胞的上清液中检测到大量VEGF（>1 ng/10(6)细胞/3天）。巨核细胞系的生长因子（白细胞介素3、血小板生成素）刺激CD41a+细胞组成性分泌VEGF。对肝素-琼脂糖富集的上清液进行蛋白质印迹分析主要检测到异构体VEGF165。此外，免疫组织化学显示多倍体巨核细胞内有细胞质VEGF。凝血酶刺激巨核细胞和血小板导致VEGF在30分钟内快速释放。我们得出结论，人巨核细胞以可诱导的方式产生和分泌VEGF。在骨髓微环境中，巨核细胞分泌的VEGF可能有助于内皮细胞的增殖。由活化血小板输送到血管损伤部位的VEGF可能启动血管生成。
