Abstract
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.
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This research was supported by The Ministry of Science, Technological Development and Innovation of the Republic of Serbia grant number 451–03-66/2024–03/200015.
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Conceptualization, V.Č., D.A. and O.M.A.; methodology, T.S., O.M.A., M.D. and D.Đ.; formal analysis, T.S., O.M.A., E.Ž. and M.D.; investigation, T.S., O.M.A., M.V., T.D. and D.Đ.; data curation, O.M.A. and M.V., writing—original draft preparation, O.M.A., T.S., V.V., and V.Č.; writing—review & editing, O.M.A. and V.Č.; visualization, O.M.A. and E.Ž.; supervision, V.Č.; project administration, V.Č. All authors have read and agreed to the published version of the manuscript.
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The study was approved by the Ethics Committee of the University Clinical Centre of Serbia, Belgrade (decision number 187/4) and the Ethics Committee of the Institute for Medical Research, Belgrade (decision number EO 117/2016).
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Mitrović-Ajtić, O., Živković, E., Subotički, T. et al. Inflammation mediated angiogenesis in chronic lymphocytic leukemia. Ann Hematol (2024). https://doi.org/10.1007/s00277-024-05781-1
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DOI: https://doi.org/10.1007/s00277-024-05781-1