Monocytes Transdifferentiate Into Lymphatic Endothelial Cells Stimulated With Endothelial Growth Factor And Inflammatory Factor

Backgrounds:Lymphangiogenesis is closely related to the development and treatment of lymph node metastasis, wound healing, secondary lymphedema and other diseases, particularly the upper limb lymphedema, which is common complication after axillary lymph node dissection. It can lead to repeated infection and upper extremity dysfunction, and seriously affect the patient's quality of life. To date, there is little ideal treatment against lymphedema. Lymphangiogenesis is undoubtedly the key point in the curing of lymphedema, among which the proliferation of lymphatic vascular endothelial cells is the most important. Lymphangiogenesis and angiogenesis are usually accompanied in many pathophysiological processes. Previous researches have demonstrated that the local infiltration of monocytes-macrophages in inflammation, cancer or immune rejection, and their number was significantly associated with lymph node metastasis, macrophages has even found incorporating into lymphatic wall, so are closely linked with the lymphangiogenesis. In addition, circulating monocytes have been proved to present some characteristics of stem cell and can transform into a variety of cells such as epithelial cells, T cells, liver cells, osteoblasts, EPCs and blood vessels endothelial cells under induction of certain factors, Moreover, monocytes are abundant and easily obtained, which has broad application prospects. However, little is known about whether monocytes can differentiate into lymphatic endothelial cells.As we all known, angiogenesis and lymphangiogenesis is often accompanied in many pathophysiological processes. The mechanisms of EPCs participating in angiogenesis have also been widely concerned. Studies have found that peripheral blood derived EPCs showed low performance of proliferation, mainly through the secretion of vascular endothelial growth factor, hepatocyte growth factor to promote angiogenesis; In addition, other researches showed that EPCs can also directly participate in angiogenesis. Then, how do the macrophages participate in lymphangiogenesis? Is directly differentiate into lymphatic endothelial cells or by secreting a series of cytokines to indirectly involved in the regulation of lymphangiogenesis? Current studies have been reported that activated macrophage could secrete VEGF-C and VEGF-D. Therefore, we suppose that monocytes may also regulate the lymphangiogenesis by secreting certain growth factors.Purpose:We planned to evaluate the possibility of transdifferentiation from monocytes to lymphatic endothelial cells in vitro and the mechanism of monocytes involved in lymphangiogenesis, and to provide theoretical basis for the therapy of mononuclear macrophages involved in Lymphedema in breast cancer after surgery.Method:Mononuclear cells were isolated from human peripheral blood by Ficoll density-gradient centrifugation, cells were induced in FN and EGM-2 in vitro. The experimental group was stimulated with100 ng/ml LPS. The expression of specific markers of lymphatic endothelial cells such as VEGFR-3, LYVE-1, Podoplanin, Porx-land the markers of endothelial cells such as vWF, CD 144 and VEGFR-2 and CD34 were detected by Immunofluorescence and RT-PCR and Flow cytometry analysis. Subsequently, cell supernatant was collected and detected the levels of VEGF-C by ELISA, further explored the mechanism involved in lymphangiogenesis.Results:(1) Monocytes isolated freshly:①By Immunocytochemistry, cells only expressed CD 14;②By RT-PCR, revealed that cells expressed the gene of VEGFR-3 and LYVE-1.③By flow cytometry, about 96.6%±0.7% cells were positive for CD 14; about 0.9%±0.2% cells were positive for both CD 14 and CD34; only 0.1%±0.1% cells were positive for CD14 and VEGFR-3; 4.08%±0.3% cells were positive for CD14 and LYVE-1; 0.2%±0.1% cells were positive for CD14 and Podoplanin. (2)Monocytes cultured in vitro for 7 days:①By immunofluorescent staining, the cells were positive for LYVE-1,Podoplanin,VEGFR-3,VEGFR-2 and vWF to varied degrees.②By RT-PCR, revealed that cells expressed the gene of VEGFR-3, Podoplanin, Prox-1, LYVE-1, vWF and CD144, while the expression of VEGFR-2 were weak positive or negative. After stimulated with LPS, the expression of the gene was increased than the control group.③Flow cytometry showed that the VEGFR-3 expression rate of EGM-2 training group cells was 1.5%±0.2%; LYVE-1 expression rate for 11.5±0.3% and the Podoplanin rate for 1.6%±0.2%. The VEGFR-3 expression rate of EGF-2 plus LPS stimulated cells was 26.9%±0.7%; LYVE-1 expression rate for 73.6%±0.3% and the Podoplanin rate for 53.1%±0.5%.④By ELISA, the level of VEGF-C in experiments group was 211.4 (179.8-437.8) pg/106 cells; the level of VEGF-C in control group was98.2 (69.9-153.1) pg/106 cells (p< 0.001).Conclusion:①Monocytes cultured in vitro for 7 days expressed both specific markers for lymphatic endothelium and common antigens of endothelial cells, indicating that monocytes could differentiate into lymphatic endothelial-like cells.②Monocytes cultured in vitro secreted VEGF-C, informed that they may participated in lymphangiogenesis through paracrine.