Identification of Vascular Lineage-Specific Genes by Transcriptional Profiling of Isolated Blood Vascular and Lymphatic Endothelial Cells

Abstract

In mammals, the lymphatic vascular system develops by budding of lymphatic progenitor endothelial cells from embryonic veins to form a distinct network of draining vessels with important functions in the immune response and in cancer metastasis. However, the lineage-specific molecular characteristics of blood vascular versus lymphatic endothelium have remained poorly defined. We isolated lymphatic endothelial cells (LECs) and blood vascular endothelial cells (BVECs) by immunomagnetic isolation directly from human skin. Cultured LECs but not BVECs expressed the lymphatic markers Prox1 and LYVE-1 and formed LYVE-1-positive vascular tubes after implantation in vivo. Transcriptional profiling studies revealed increased expression of several extracellular matrix and adhesion molecules in BVECs, including versican, collagens, laminin, and N-cadherin, and of the growth factor receptors endoglin and vascular endothelial growth factor receptor-1/Flt-1. Differential immunostains of human skin confirmed the blood vessel-specific expression of these genes. During embryonic development, endoglin expression was gradually down-regulated on lymphatic endothelium whereas vascular endothelial growth factor receptor-1 was absent from lymphatics. We also identified several genes with specific expression in LECs. These results demonstrate that some lineage-specific genes are only expressed during distinct developmental stages and they identify new molecular markers for blood vascular and lymphatic endothelium with im- The lymphatic system consists of a vascular network of thin-walled capillaries that drain protein-rich lymph from the extracellular spaces within most organs and that play major roles in the immune response and in tumor metastasis.1,2 Lymphatic vessels provide the conduit for antigen-presenting cells from the organ exposed to antigens to the regional lymph nodes, involving active recruitment of antigen-presenting cells by chemokines and other mediators secreted by lymphatic endothelium.3 Moreover, the early dissemination of malignant tumors frequently occurs via lymphatic vessels to regional lymph nodes, and the recent discovery of active tumor lymphangiogenesis and its role in cancer metastasis has drawn considerable attention to the molecular mechanisms that control activation and proliferation of lymphatic endothelium. 2,4

Low podoplanin expression in pretreatment biopsy material predicts poor prognosis in advanced-stage squamous cell carcinoma of the uterine cervix treated by primary radiation

“…[20][21][22] Antibodies to podoplanin, including D2-40, were reported to be selective markers for lymphatic endothelium and to be useful in identifying the presence of lymphatic invasion in various malignant neoplasms, including cervical carcinoma. [15][16][17] However, little is known about the biological function of podoplanin.…”

Development of the lymphatic vascular system: A mystery unravels

“…These cells maintain their lineage-specific differentiation even after several passages in vitro. Comparative microarray analyses of their specific transcriptomes revealed that the majority of all genes investigated (approximately 98%) were expressed at comparable levels by the two endothelial cell types (Petrova et al, 2002;Hirakawa et al, 2003), corroborating their close genetic relationship. Whereas the specific function of many of the differentially expressed genes still remains elusive, several molecules have been studied in genetic mouse models and have been found to play important roles in lymphatic development and function (Table 1).…”

“…Major advances in lymphatic research have been enabled by the recent establishment of defined cultures of blood vascular and lymphatic endothelial cells isolated from human skin (Kriehuber et al, 2001;Makinen et al, 2001b;Podgrabinska et al, 2002;Hirakawa et al, 2003). These cells maintain their lineage-specific differentiation even after several passages in vitro.…”

“…VEGFR-2, also known as KDR or Flk1, is a receptor for VEGF-A, VEGF-C, and VEGF-D, and is expressed by blood vascular and lymphatic endothelial cells in situ and in vitro (Kriehuber et al, 2001;Hirakawa et al, 2003;. Although the role of VEGFR-2 in angiogenesis has been well established, the possible contribution of VEGF-A signaling through VEGFR-2 to lymphangiogenesis is currently a matter of controversy.…”

“…Podoplanin is a mucin-type transmembrane glycoprotein that is expressed by lymphatic, but not by blood vascular endothelial cells in vivo and in vitro (Wetterwald et al, 1996;Kriehuber et al, 2001;Petrova et al, 2002;Hirakawa et al, 2003;Schacht et al, 2003). In addition, podoplanin is expressed by several other cell types, including type I alveolar cells of the lung, cells of the choroid plexus, ciliary epithelial cells of the eye, osteocytes and kidney podocytes, and multiple synonyms have been used in the literature, such as RTI40, murine gp38, canine gp40, human gp36, and murine PA2.26 Schacht et al, 2003).…”