Isolation and characterization of lymphatic microvascular endothelial cells from human tonsils.
Human lymphatic endothelial cells (LECs) have isolated prevalently from human derma and tumors. As specialized lymphatic organs within the oropharynx, palatine tonsils are easily obtained and rich in lymphatic venules. Using a two-step purification method based on the sorting of endothelial cells with Ulex Europaeus Agglutinin 1 (UEA-1)-coated beads, followed by purification with monoclonal antibody D2-40, we successfully purified LECs from human palatine tonsils. The LECs were expanded on flasks coated with collagen type 1 and fibronectin for up to 8-10 passages and then analyzed for phenotypic and functional properties. Cultured cells retained the phenotypic pattern of the lymphatic endothelium of palatine tonsils and expressed functional VEGFR-3 molecules. In fact, stimulation with VEGFR-3 ligand, the vascular endothelium grow factor C, induced a marked increase in cell proliferation. Similarly to blood endothelial cells (BECs), LECs were able to form tube-like structure when seeded in Cultrex basement membrane extract. Comparative studies performed on LECs derived from palatine tonsils and iliac lymphatic vessels (ILVs), obtained with the same procedures, showed substantial discrepancies in the expression of various lymphatic markers. This points to the existence of micro- and macrovessel-derived LECs with different phenotypes, possibly involving different biological activities and functions. Palatine tonsil- and ILV-derived LECs may, therefore, represent new models for investigating function and biochemical properties of these lymphatic endothelia.
Garrafa
Emirena
E
Department of Microbiology, University of Brescia, Brescia, Italy. emirenagarrafa@libero.it
Alessandri
Giulio
G
Benetti
Anna
A
Turetta
Daniela
D
Corradi
Attilio
A
Cantoni
Anna Maria
AM
Cervi
Edoardo
E
Bonardelli
Stefano
S
Parati
Eugenio
E
Giulini
Stefano Maria
SM
Ensoli
Barbara
B
Caruso
Arnaldo
A
D000911:Antibodies, Monoclonal / Q000737:chemistry; D058846:Antibodies, Monoclonal, Murine-Derived; D018929:Cell Culture Techniques / Q000379:methods; D049109:Cell Proliferation / Q000187:drug effects; D002469:Cell Separation; D042783:Endothelial Cells / Q000737:chemistry / Q000166:cytology* / Q000187:drug effects; D006023:Glycoproteins / Q000032:analysis; D018398:Homeodomain Proteins / Q000032:analysis; D006801:Humans; D019394:Ki-67 Antigen / Q000032:analysis; D042601:Lymphatic Vessels / Q000166:cytology; D008562:Membrane Glycoproteins / Q000032:analysis; D014066:Palatine Tonsil / Q000737:chemistry / Q000166:cytology*; D037121:Plant Lectins / Q000737:chemistry; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000032:analysis; D013194:Staining and Labeling; D025521:Tumor Suppressor Proteins; D014470:Umbilical Cord / Q000166:cytology; D042582:Vascular Endothelial Growth Factor C / Q000494:pharmacology; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000032:analysis; D033921:Vesicular Transport Proteins; D014841:von Willebrand Factor / Q000032:analysis; D000098551:Prospero-Related Homeobox 1 Protein; D000098853:Podoplanin
D000911:Antibodies, Monoclonal; D058846:Antibodies, Monoclonal, Murine-Derived; D006023:Glycoproteins; D018398:Homeodomain Proteins; D019394:Ki-67 Antigen; D008562:Membrane Glycoproteins; D037121:Plant Lectins; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D025521:Tumor Suppressor Proteins; D042582:Vascular Endothelial Growth Factor C; D040301:Vascular Endothelial Growth Factor Receptor-2; D033921:Vesicular Transport Proteins; D014841:von Willebrand Factor; D000098551:Prospero-Related Homeobox 1 Protein; C119248:LYVE1 protein, human; C117468:PDPN protein, human; D000098853:Podoplanin; C036850:Ulex europaeus lectins; C500987:monoclonal antibody D2-40