Bruce Rappaport Faculty of Medicine, Technion, Israel Institute of Technology, Haifa, Israel.
Department of Hematology and Bone Marrow Transplantation, Rambam Health Care Campus, 7, Ha'Aliya St., Haifa, 3109601, Israel.
Angiogenesis. 2019 Feb;22(1):185-196. doi: 10.1007/s10456-018-9649-y. Epub 2018 Nov 1.
Bone marrow microenvironment is known to support angiogenesis, thus contributing to progression of multiple myeloma (MM). Bortezomib, a proteasome inhibitor (PI) widely used in MM treatment, has anti-angiogenic activity. Extracellular vesicles (EVs), shedding from cell surface, serve as mediators in cell-to-cell communication. We have hypothesized that MM cells (MMCs) treated with bortezomib generate EVs that could diminish angiogenesis, thus limiting MM progression. In the present study, EVs were obtained from MMCs (RPMI-8226), untreated (naïve) or pre-treated with bortezomib. EVs were outlined using NanoSight, FACS, protein arrays and proteasome activity assays. The impact of MMC-EVs on endothelial cell (EC) functions was assessed, employing XTT assay, Boyden chamber and Western blot. A high apoptosis level (annexin V binding 70.25 ± 16.37%) was observed in MMCs following exposure to bortezomib. Compared to naïve EVs, a large proportion of bortezomib-induced EVs (Bi-EVs) were bigger in size (> 300 nm), with higher levels of annexin V binding (p = 0.0043).They also differed in content, presenting with increased levels of pro-inflammatory proteins, reduced levels of pro-angiogenic growth factors (VEGFA, PDGF-BB, angiogenin), and displayed lower proteasome activity. Naïve EVs were found to promote EC migration and proliferation via ERK1/2 and JNK1/2/3 phosphorylation, whereas Bi-EVs inhibited these functions. Moreover, Bi-EVs appeared to reduce EC proteasome activity. EVs released from apoptotic MMCs following treatment with bortezomib can promote angiogenesis suppression by decreasing proliferation and migration of EC. These activities are found to be mediated by specific signal transduction pathways.
骨髓微环境已知可支持血管生成，从而促进多发性骨髓瘤（MM）的进展。硼替佐米是一种广泛用于 MM 治疗的蛋白酶体抑制剂（PI），具有抗血管生成活性。细胞表面脱落的细胞外囊泡（EVs）可作为细胞间通讯的介质。我们假设用硼替佐米处理的 MM 细胞（MMCs）产生的 EVs 可减少血管生成，从而限制 MM 的进展。在本研究中，从未经处理（原始）或用硼替佐米预处理的 MM 细胞（RPMI-8226）中获得 EVs。使用 NanoSight、FACS、蛋白质阵列和蛋白酶体活性测定法描绘 EVs。通过 XTT 测定法、Boyden 室和 Western blot 评估 MMC-EVs 对内皮细胞（EC）功能的影响。硼替佐米暴露后，MMC 中观察到高凋亡水平（膜联蛋白 V 结合 70.25 ± 16.37%）。与原始 EVs 相比，硼替佐米诱导的 EVs（Bi-EVs）中较大比例（>300nm）的 EVs 尺寸较大，膜联蛋白 V 结合水平较高（p=0.0043）。它们在内容上也有所不同，表现出更高水平的促炎蛋白，更低水平的促血管生成生长因子（VEGFA、PDGF-BB、血管生成素），并且蛋白酶体活性较低。发现原始 EVs 通过 ERK1/2 和 JNK1/2/3 磷酸化促进 EC 迁移和增殖，而 Bi-EVs 抑制这些功能。此外，Bi-EVs 似乎降低了 EC 的蛋白酶体活性。硼替佐米处理后凋亡 MMC 释放的 EVs 通过降低 EC 的增殖和迁移来促进血管生成抑制。这些活性被发现是由特定的信号转导途径介导的。
