VEGF-PKD1-HDAC7Axis Regulates Circulating Endothelial Progenitor Cells Involving In Non-Hodgkin’s Lymphoma Angiogenesis

Part OneThe circulating EPCs in angiogenensis of NHLObjective:To establish a method in isolation, culture and identification of primary endothelial progenitor cells (EPCs) in vitro; and to study the role of EPCs in lymphoma angiogenesis.Methods:We cultured EPCs form human umbilical cord blood mononuclear cells, and amplificated the. cells by the EGM-2MV medium in vitro. We used cell function assay, cell surface marker to identificate EPCs. Non-Hodgkin’s lymphoma subcutaneous transplantation tumor models were established and107human GFP-labeled EPCs were intravenous infused into nude mice tail vein when the tumor mass expand to1cm3. We did this assay to understand whether EPCs can recruit into lymphoma and their specific location. We aslo co-cultured EPCs and mature endothelial cells to study the change of vascular formation ability.Results:EPCs were separated from human umbilical cord blood mononuclear cells. Colonies of endothelial cells showed up in the7days, then expanded everyday and identified as monolayers of cobblestone appearing cells. EPCs were identified by examining the morphology, the function of angiogenesis and by uptaking of acetylated low-density lipoprotein (red) and binding to Lectin (green) like the other endothelial cells. HUVECs and EAhy.926cells also showed the same characters. EPCs labeled with CD34, CD133, and CD309(VEGFR-2) antibodies were tested by Flow Cytometry. We found that the cells initially expressed hematopoietic stem cells markers, like CD133and CD34, but expressed little endothelial cell markers, like CD309. After2weeks culture, the cells gradually lost the stem cells markers but expressed endothelial cells marker. Endothelial progenitor cells injected into nude mice in vivo, in the3days, the10days after injection, The human GFP-labeled EPCs were green and located around the vessels at all check points. To visualize blood vessels in the tumor tissue of lymphoma, frozen sections were subjected to CD31staining. Red fluorescence indicates CD31+capillaries. The fluorescent intensity increased by time after the infusion of EPCs. The co-cultured group had higher capacity of tube formation than the HUVECs group both in the total tube length and tube formation areas. The wound-healing assay showed that the co-cultured group migrated longer distance than the HUVEC group in the same timeConclusion:1. We can successfully cultivate the primary EPCs from umbilical cord blood mononuclear cells in vitro;2. EPCs can be recruited to the lymphoma transplanted tumor and participate in angiogenesis; and endothelial progenitor cells can promote lymphoma angiogenesis;3. Co-cultured EPCs and mature endothelial cells could promote lymphoma angiogenesis by promoting cell migration and tube formation ability. Part Two NHL microenvironment promote angiogenesis of endothelial progenitor cells by activating HDAC7 Objective:To study the lymphoma microenvironment on the angiogenesis of endothelial progenitor regulated by HDAC7protein.Methods:We used EPCs as the experimental cells, non-Hodgkin’s lymphoma cells supernatant and VEGF as intervention factors. We study the effect of NHL supernatant on EPCs by tube formation assay, and detected the effect of NHL supernatant on HDAC7protein expression in EPCs by Western bloting. Then we used siRNA technology to silence HDAC7gene in EPCs, and to test the effect of the HDAC7silence on the angiogenesis ability (migration and tube formation) of EPCs.Results:Lymphoma cells supernatant could promote the tube formation ability of EPCs, and could activate phosphorylation of HDAC7protein. HDAC7genes silence of EPCs can weaken the cells migration and tube formation ability.Conclusion:Lymphoma microenvironment can promote tube formation of EPCs by activation of HDAC7protein, so as to enhance cells angiogenesis. Part ThreeMechanisms of the VEGF-PKD1-HDAC7signaling pathways regulating endothelial progenitor cellsObjective:To study VEGF how to regulate the expression of HDAC7protein, and to explore the possible activation pathway.Method:We used Western blotting and immunofluorescence technique to study the activation of HDAC7protein and HDAC7intracellular localization. At the same time, we used VEGF downstream kinases inhibitors to inhibit the expression of kinase downstream respectively, and used western blotting and immunofluorescence technique to study the activation of HDAC7protein and HDAC7intracellular localization.Result:VEGF-A treatment induced HDAC7phosphorylation quickly within10minutes, and the activation reached a maximum at30minutes and gradually decreased after60 minutes. HDAC7localization in EPCs was analyzed by fluorescence microscope. Labeled-HDAC7located mainly in the nuclear in the absence of the simulation. In response to VEGF-A, labeled-HDAC7in EPCs showed nucleocytoplasmic shuttling. HDAC7nuclear export first turned up at30minutes following VEGF-A treatment, then proportion of cells with HDAC7cytoplasmic accumulation increased by time and reached a maximum of80%after2hours of treatment. Labeled-HDAC7then gradually relocated in the nucleus and primarily went back to the nucleus after24hours of VEGF-A simulation. To determine which kinase can phosphorylate HDAC7in response to VEGF-A, EPCs were pretreated with kinase inhibitors before VEGF-A treatment. When EPCs were pretreated with PKD1inhibitor Go6976at30minutes before VEGF-A treatment, HDAC7phosphorylation was obviously inhibited in dose-dependent manner. Neithor PI3K inhibitor LY294002nor MEK1/2inhibitor U0126had any effect on VEGF-A induced HDAC7phosphorylation. G66976also inhibited the nucleocytoplasmic shuttling of HDAC7induced by VEGF-A.Conclusion:VEGF induced an increase of p-HDAC7and nuclear export in a time-dependent manner, which was partly inhibited by PKD1inhibitor, but not by PI3K inhibitor or MEK inhibitor.