Department of Molecular Oncology, Institute for Medical Research, National Institute of Republic of Serbia, University of Belgrade, Dr. Subotića 4, 11129, Belgrade, Serbia.
Lymphoma Center, Clinic for Hematology, University Clinical Center of Serbia, Belgrade, Serbia.
Ann Hematol. 2024 Aug;103(8):2865-2875. doi: 10.1007/s00277-024-05781-1. Epub 2024 May 7.
Chronic inflammation has been identified in leukemias as an essential regulator of angiogenesis. B-chronic lymphocytic leukemia (CLL) cells secrete high levels of vascular endothelial growth factor (VEGF) and hypoxia inducible factor 1 alpha (HIF1α). The aim was to assess the role of inflammation in activation of angiogenic factors: endothelial nitric oxide synthase (eNOS), HIF1α and VEGF via proliferation related signaling pathways and VEGF autocrine control. We isolated mononuclear cells (MNC) and CD19 cells from peripheral blood of 60 patients with CLL. MNC were treated with pro-inflammatory interleukin-6 (IL-6) and VEGF, in combination with inhibitors of JAK1/2 (Ruxolitinib), mTOR (Rapamycin), NF-κB (JSH23), SMAD (LDN-193189) and PI3K/AKT (Ly294002) signaling pathways, to evaluate eNOS, VEGF and HIF1α expression by immunoblotting, immunocytochemistry and RT-qPCR. Also, we investigated IL-6 dependent neovascularization in human microvascular endothelial cells (HMEC-1) in co-culture with MNC of CLL. The angiogenic factors eNOS, VEGF and HIF1α had significantly higher frequencies in MNC of CLL in comparison to healthy controls (p < 0.001) and CD19 cells of CLL. IL-6 increased the quantity of HIF1α (p < 0.05) and VEGF positive cells in the presence of JSH23 (p < 0.01). VEGF increased HIF1α (p < 0.05), and decreased eNOS gene expression (p < 0.01) in MNC of CLL. VEGF significantly (p < 0.001) increased the number of HIF1α positive MNC of CLL, prevented by inhibitors of JAK1/2, PI3K and mTOR signaling pathways. VEGF stimulation of SMAD (p < 0.05) and STAT5 (p < 0.01) signaling has been prevented by inhibitors of JAK1/2, mTOR, PI3K and SMAD signaling, individually (p < 0.01) or mutually (p < 0.001). Also, we showed that MNC of CLL and IL-6 individually stimulate neovascularization in co-culture with HMEC-1, without a cumulative effect. We demonstrated elevated angiogenic factors in CLL, while VEGF and IL-6 independently stimulated HIF1α. VEGF stimulation of HIF1α was mostly mTOR dependent, while IL-6 stimulation was NF-κB dependent.
慢性炎症已被确定在白血病中是血管生成的重要调节因子。B-慢性淋巴细胞白血病（CLL）细胞分泌高水平的血管内皮生长因子（VEGF）和缺氧诱导因子 1α（HIF1α）。目的是评估炎症在激活血管生成因子方面的作用：内皮型一氧化氮合酶（eNOS）、HIF1α 和 VEGF 通过增殖相关信号通路和 VEGF 自分泌控制。我们从 60 例 CLL 患者的外周血中分离单核细胞（MNC）和 CD19 细胞。MNC 用促炎白细胞介素 6（IL-6）和 VEGF 处理，并用 JAK1/2（芦可替尼）、mTOR（雷帕霉素）、NF-κB（JSH23）、SMAD（LDN-193189）和 PI3K/AKT（Ly294002）信号通路抑制剂处理，通过免疫印迹、免疫细胞化学和 RT-qPCR 评估 eNOS、VEGF 和 HIF1α 的表达。此外，我们还研究了在与 CLL 的 MNC 共培养的人微血管内皮细胞（HMEC-1）中 IL-6 依赖性新生血管形成。与健康对照组（p<0.001）和 CLL 的 CD19 细胞相比，CLL 的 MNC 中血管生成因子 eNOS、VEGF 和 HIF1α 的频率明显更高。IL-6 在存在 JSH23 的情况下增加了 HIF1α（p<0.05）和 VEGF 阳性细胞的数量。VEGF 增加了 CLL 的 MNC 中的 HIF1α（p<0.05），并降低了 eNOS 基因表达（p<0.01）。VEGF 显著（p<0.001）增加了 CLL 的 MNC 中 HIF1α 阳性细胞的数量，这可以被 JAK1/2、PI3K 和 mTOR 信号通路的抑制剂所阻止。VEGF 刺激 SMAD（p<0.05）和 STAT5（p<0.01）信号通路可分别（p<0.01）或相互（p<0.001）被 JAK1/2、mTOR、PI3K 和 SMAD 信号通路的抑制剂所阻止。此外，我们还表明 CLL 的 MNC 和 IL-6 单独刺激与 HMEC-1 的共培养中的新生血管形成，而没有累积效应。我们证明了 CLL 中升高的血管生成因子，而 VEGF 和 IL-6 独立地刺激 HIF1α。VEGF 刺激 HIF1α 主要依赖于 mTOR，而 IL-6 刺激依赖于 NF-κB。
