Monocytes are an important proportion of cells in peripheral blood, which migrate to local tissues under the condition of tumor, wound or inflammation, and differentiate into macrophages. In these pathological processes, various cells groups including macrophages produce FN, TNF-α and VEGF-C, which may exert certain effects on macrophages transdifferentiating into lymphatic endothelial cells. A study on murine cornea demonstrated that newly formed lymphatic vessels in macrophage infiltrating area were not continuous with limbal existing lymphatic vessels, but were closely related to CD11b+ macrophages in that area[15]. Thus, monocytes may have the potential to transdifferentiate into lymphatic endothelial cells, which is yet to be proved in vitro condition.
The monocytes were obtained as described[16]. Firstly, several specific markers of lymphatic endothelial cells on freshly collected monocytes from healthy donors were examined. The expression of LYVE-1 was positive while the expression of Podoplanin, Porx-1 and VEGFR-3 as well as common antigens of endothelial cells vWF, eNOS andVEGFR-2 turned out to be negative. Subsequently, macrophage expression of these markers was tested under different culture conditions. Following 24 hours incubation, Podoplanin, Porx-1 and VEGFR-3 were positive detected by RT-PCR in FN-plated group and in TNF-α treated group. However, the expression of vWF, eNOS and VEGFR-2 remained negative. By immunocytochemistry, the expression of Podoplanin, Porx-1, and VEGR-3 was positive, while vWF, eNOS and VEGFR-2 was negative. According to previous researches, monocytes will transdifferentiate into vascular endothelial cells under the stimulation of growth factors including VEGF[7-9]. Thus, we suppose that the lack of vWF, eNOS and VEGFR-2 was due to insufficient amount of VEGF or inadequate induction period.
VEGFR-3 is a lymphatic-specific marker[17-18]. As it is the receptor of VEGF-C which is a lymphatic-specific growth factor, VEGFR-3 plays an important role in the lymphangiogenesis. In tumor or inflamed condition, local macrophages become a crucial resource of VEGF-C[19]. In addition, both VEGF-C and its receptor VEGFR-3 have been reported to be produced and expressed by activated monocytes[20]. In this study, stimulated with FN or TNF-α, monocytes expressed VEGFR-3. The expression of VEGFR-3 is important for monocytes differentiating into lymphatic endothelial cells.
Fibronectin is a high-molecular weight glycoprotein that distributes extensively in extracellular matrix. It is produced by various cells types including fibroblasts, monocytes and tumor cells, and its production increases in inflamed condition. Usually, FN is used as an adhesive in monocyte and neurocyte culturing. In recent years, however, FN is discovered to play a crucial role in migration and differentiation of cells and tissue repairing[21]. We proved that with the induction of FN, monocytes expressed Podoplanin, Porx-1 and VEGFR-3, but the mechanism is yet to be discovered.
TNF-α is one of the most common inflammatory factors produced by various cells types. By inducing monocytes with TNF-α, Podoplanin, Porx-1 and VEGFR-3 were positively expressed. Previous study proved that TNF-α induced macrophages in tumor stroma to produce VEGF-C[22], while TNF-α upregulated the VEGFR-3 expression simultaneously[23]. Therefore, TNF-α inducing the expression of lymphatic endothelial markers may be mediated by VEGFR-3 pathway.
In summary, monocytes expressed lymphatic endothelial markers in a short period of stimulation with TNF-α and FN, while the expression of endothelial common markers was negative. It is supposed that monocytes may completely transdifferentiate into lymphatic endothelial cells with sufficient amount of endothelial growth factors such as VEGF. Considering the scarcely distributed endothelial progenitor cells in peripheral blood, monocytes may be an ideal resource of lymphatic endothelial progenitor cells, because they are more abundant in amount and easy to collect, which is of great value in clinical use.
