Constitutive production and thrombin-induced release of vascular endothelial growth factor by human megakaryocytes and platelets
Constitutive production and thrombin-induced release of vascular endothelial growth factor by human megakaryocytes and platelets
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DOI：
10.1073/pnas.94.2.663
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发表时间：
1997-01-21
影响因子：
11.1
通讯作者：
Rafii, S
中科院分区：
文献类型：
--
作者：
Mohle, R;Green, D;Rafii, S
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的增殖和分化。在这些长期培养中，骨髓微血管内皮细胞单层在缺乏外源性内皮生长因子的情况下保持其细胞完整性。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血管内皮生长因子（VEGF）的情况。用kit配体和血小板生成素将造血祖细胞体外扩增10天，生成巨核细胞CD41a(+)，并用免疫磁微珠进一步纯化。通过逆转录- pcr，我们发现巨核细胞系（Dami、HEL）和纯化的巨核细胞表达了三种VEGF亚型（121、165和189个氨基酸）的mRNA。在骨髓分离的Dami细胞、离体巨核细胞和CD41a（+）细胞的上清液中检测到大量的VEGF （>.1 ng/10(6)个细胞/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，富集肝素- sepharose的上清Western blotting主要检测同种异构体VEGF（165）。此外，免疫组织化学显示多倍体巨核细胞中存在胞浆内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，人类巨核细胞以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可能促进内皮细胞的增殖。血管内皮生长因子通过活化的血小板传递到血管损伤部位，可以启动血管生成。
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 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 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 VEGF(165). 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.