Molecular Cardiology Podoplanin-Expressing Cells Derived From Bone Marrow Play a Crucial Role in Postnatal Lymphatic Neovascularization

Background—Emerging evidence has suggested a contribution of bone marrow (BM) cells to lymphatic vessel formation; however, the exact phenotype of the cells with lymphatic endothelial progenitor cell function has yet to be identified. Here, we investigate the identity of BM-derived lymphatic endothelial progenitor cells and their role in lymphatic neovascularization. Methods and Results—Culture of BM-mononuclear cells in the presence of vascular endothelial growth factors A and C and endothelial growth factor resulted in expression of lymphatic endothelial cell markers. Among these cells, podoplanin ϩ cells were isolated by magnetic-activated cell sorting and characterized by fluorescence-activated cell sorter analysis and immunocytochemistry. These podoplanin ϩ cells highly express markers for lymphatic endothelial cells, hematopoietic lineages, and stem/progenitor cells; on further cultivation, they generate lymphatic endothelial cells. We further confirmed that podoplanin ϩ cells exist in small numbers in BM and peripheral blood of normal mice but are significantly (15-fold) augmented on lymphangiogenic stimuli such as tumor implantation. Next, to evaluate the potential of podoplanin ϩ cells for the formation of new lymphatic vessels in vivo, we injected culture-isolated or freshly isolated BM-derived podoplanin ϩ cells into wound and tumor models. Immunohistochemistry demonstrated that the injected cells were incorporated into the lymphatic vasculature, displayed lymphatic endothelial cell phenotypes, and increased lymphatic vascular density in tissues, suggesting lymphvasculogenesis. Podoplanin ϩ cells also expressed high levels of lymphangiogenic cytokines and increased proliferation of lymphatic endothelial cells during coculture, suggesting a lymphangiogenic or paracrine role. Conclusions—Our results provide compelling evidence that BM-derived podoplanin ϩ cells, a previously unrecognized cell type, function as lymphatic endothelial progenitor cells and participate in postnatal lymphatic neovascularization through both lymphvasculogenesis and lymphangiogenesis. L ymphatic vessels play an important role in the pathogen-esis of diseases such as lymphedema and tumors, 1,2 and an understanding of lymphatic biology is crucial to develop strategies to prevent or treat these diseases. Investigation of lymphatic vascular growth was made possible by the discovery of specific markers for lymphatic endothelial cells (LECs). The establishment of the lymphatic vasculature during mouse embryogenesis begins with expression of homeobox transcriptional factor PROX-1 in a subset of the venous endothelial cells of the cardinal vein 3 that express vascular endothelial growth factor (VEGF) receptor (VEGFR)-3 4 and LYVE-1. 5 Subsequently, the PROX-1– expressing (PROX-1 ϩ) cells migrate out to form the primary lymphatic plexus, which undergoes further remodeling to form a mature network of LECs that express other LEC markers such …