Isolation and characterization of dermal lymphatic and blood endothelial cells reveal stable and functionally specialized cell lineages

A plexus of lymphatic vessels guides interstitial fluid, passenger leukocyt es, and tumor cells toward regional lymph nodes. Microvascular endothelial cells (ECs) of lymph channels (LECs) are difficult to distinguish from thos e of blood vessels (BECs) because both express a similar set of markers, su ch as CD31, CD34, podocalyxin, von Willebrand factor (vWF), etc. Analysis o f the specific properties of LECs was hampered so far by lack of tools to i solate LECs. Recently, the 38-kD mucoprotein podoplanin was found to be exp ressed by microvascular LECs but not BECs in vivo. Here we isolated for the first time podoplanin(+) LECs and podoplanin(-) BECs from dermal cell susp ensions by multicolor flow cytometry. Both EC types were propagated and sta bly expressed VE-cadherin, CD31, and vWF. Molecules selectively displayed b y LECs in vivo, i.e., podoplanin, the hyaluronate receptor LYVE-1, and the vascular endothelial cell growth factor (VEGF)-C receptor, fins-like tyrosi ne kinase 4 (Flt-4)/VEGFR-3, were strongly expressed by expanded LECs, but not BECs. Conversely, BECs but not LECs expressed VEGF-C. LECs as well as B ECs formed junctional contacts with similar molecular composition and ultra structural features. Nevertheless, the two EC types assembled in vitro in v ascular tubes in a strictly homotypic fashion. This EC specialization exten ds to the secretion of biologically relevant chemotactic factors: LECs, but not BECs, constitutively secrete the CC chemokine receptor (CCR)7 ligand s econdary lymphoid tissue chemokine (SLC)/CCL21 at their basal side, while b oth subsets, upon activation, release macrophage inflammatory protein (MIP) -3 alpha /CCL20 apically. These results demonstrate that LECs and BECs cons titute stable and specialized EC lineages equipped with the potential to na vigate leukocytes and, perhaps also, tumor cells into and out of the tissue s.