Potential Of Monocyte-derived Multipotential Cells Transdifferentiating Into Lymphatic Endothelial Cells

Backgrounds:To date, there is no ideal treatment against lymphedema. Especially in recent years, the incidence of breast cancer increased year by year, more and more patients endured upper limb lymphedema after breast cancer radical surgery subsequently. Lacking effective treatment, it may develop into edema and sclerosis locally or even in the whole limb. Lymphangiogenesis is undoubtedly the key point in the curing of lymphedema, among which the proliferation of lymphatic vascular endothelial cells is the most important. Previous researches have demonstrated that monocytes and macrophages perform the function of pro-lymphangiogenesis by secreting VEGF-C/D or directly integrating into lymphatic vessels. Furthermore, circulating monocytes have been proved to present some characteristics of stem cell and can transform into endothelial progenitor cells which may transdifferentiate into vascular endothelial cells under induction of certain factors such as VEGF. Therefore, using monocytes-derived endothelial for the purpose of angiogenesis or treating ischemic disease has become a hot spot in recent years. However, there has been no evidence that monocytes derived endothelial progenitor cells can transdifferentiate into lymphatic endothelial cells. Masataka K et al successfully induced peripheral CD14+ monocytes into MOMCs, which differentiated into osteocytes, myocytes, chondrocytes, adipocytes, endothelial cells and etc. Under stimulation of various cytokines. But whether monocytes derived endothelial cells obtain biological characteristics of lymphatic vascular endothelial cells is yet unknown. In our experiment, we follow the method of Masataka K et al. MOMCs were isolated, cultured and stimulated. Common endothelial markers, VEGFR-2, CD144, vWF and CD34, and specific lymphatic endothelial markers, VEGFR-2, CD144, vWF and CD34 were examined on both proteic and transcriptional level by the method of immunocytofluorescence and RT-PCR.Purpose:We plan to test the possibility of monocytes transdifferentiating into lymphatic endothelial cells in vitro, which provides the research of lymphangiogenesis with further evidence.Method:Fresh peripheral blood was obtained from healthy donors and mononuclear cells were isolated. Incubated in 10%FBS supplemented L-DMEM for 8-12 h, the adhering cells were collected and examined by the method of flow cytometry and RT-PCR; mononuclear cells obtained were inoculated to FN pre-coated wells and cultured in 10%FBS supplemented L-DMEM for 7-10 d after which they transformed into MOMCs. lOng/ml of TNF-a was supplemented to the stimulation group which was chosen randomly 1 d before the cells were harvested. We used the method of immumocytofluorescence and RT-PCR to detect specific markers for lymphatic endothelium and common antigens of endothelial cells on both the proteinic and transcriptional level. Subsequently, MOMCs with or without stimulation of TNF-a were inoculated into EGM-2 and examined after 3 or 7 d of culturing.Results:By flow cytometry,96% of adhering cells cultured for 8-12 h turned out to be CD 14+ monocytes. RT-PCR revealed that the cells were positive for VEGFR-3, but the expression of LYVE-1, Podoplanin, Prox1, vWF, CD144, VEGFR-2 and CD34 were all negative. By RT-PCR, MOMCs expressed the Mrna of VEGFR-3, Podoplanin, Prox-1, CD34 and vWF, while the expression of LYVE-1, CD144 and VEGFR-2 were negative. By immunofluorescent staining, the cells were positive for VEGFR-3 and VEGFR-2. Treated with TNF-αfor 24 h, the cells showed no substantial difference from the non-treated cells. After 3 d of culturing in EGM-2, the cells were positive for LYVE-1, Podoplanin, Prox-1, CD34, CD144, VEGFR-2 and vWF, but negative for VEGFR-3 by RT-PCR. By immunofluorescenct staining, the cells were positive for VEGFR-3, LYVE-1, Podoplanin, VEGFR-2 and vWF to varied degrees. At the end of Day 7, the cells were examined positive for Mrna of LYVE-1, Podoplanin, Prox-1, CD34 and vWF, but on a lower level compared to the result of Day 3. The expression of VEGFR-3, CD144 and VEGFR-2 were negative. After 3 d of culturing, TNF-αpre-treated MOMCs were positive for Podoplanin, Prox-1 and CD34 by RT-PCR to a smaller extent compared to the non-treated group. The expression of VEGFR-3, LYVE-1, CD144, VEGFR-2 and vWF were all negative.Conclusion:The cells used in experiment were mostly CD 14+ monocytes. By stimulating MOMCs with FN and EGM-2 simultaneously, the cells expressed both common antigens of endothelial cells and specific markers for lymphatic endothelium, indicating that MOMCs could differentiate into lymphatic endothelial-like cells. The expression of VEGFR-2 and VEGFR-3 of MOMCs and MOMCs derived endothelial-like cells are temporal and instable, then the cells could not proliferate.