Development and characterization of endothelial cells from rat microlymphatics.
BACKGROUND
The lymphatic endothelium is important to the functioning of the lymphatic system, including lymphatic remodeling, control of vessel tone, and lymphatic movement of fluids, macromolecules, and cells. Many of these events occur principally at the level of the microlymphatics. To evaluate the role of the microlymphatic endothelium, a suitable cultured cell line would be useful. We have developed a technique to isolate and culture endothelial cells from microscopic lymphatics, approximately 100 microm in diameter.


RESULTS
To isolate the rat mesenteric lymphatic endothelial cells (RMLEC), the rat was anesthetized and the mesentery carefully exteriorized. A suitable microlymphatic was located and carefully microdissected from the surrounding mesentery. The vessel was carefully cleaned, cannulated, everted, and then incubated on a gelatin-coated plastic culture dish until small patches of cells migrated off of the vessel (3-4 days later.) The explanted vessel was then removed. The remaining cells were cultured and screened for endothelial phenotype. Nonendothelial cells were destroyed. The endothelial nature of the remaining cells was verified by: 1) morphology, 2) uptake of fluorescent acetylated-LDL, 3) staining for von Wille-brand factor, PECAM-1, ecNOS, LYVE-1, VEGFR-3, and 4) essentially negative alpha-vascular smooth muscle actin staining. The defined RMLEC were passed and the profile of adhesion molecules present on the RMLEC was then determined using PCR and immunofluorescence.


CONCLUSIONS
We developed and partially characterized a line of cultured microlymphatic endothelium. RMLEC express known endothelial- and lymphatic-specific markers as well as the following adhesion molecules: N-cadherin, E-cadherin, PECAM-1, alpha-catenin, beta-catenin, gamma-catenin, p120, and a variety of integrins.
Hayes
H
H
Department of Medical Physiology, Cardiovascular Research Institute, The Texas A&M University System Health Science Center, College Station, Texas, 77843-1114, USA.
Kossmann
E
E
Wilson
E
E
Meininger
C
C
Zawieja
D
D
D000199:Actins / Q000096:biosynthesis; D000818:Animals; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000378:metabolism; D017931:DNA Primers / Q000494:pharmacology; D042783:Endothelial Cells / Q000473:pathology*; D004729:Endothelium, Lymphatic / Q000166:cytology*; D006023:Glycoproteins / Q000096:biosynthesis; D007150:Immunohistochemistry; D016023:Integrins / Q000378:metabolism; D008077:Lipoproteins, LDL / Q000378:metabolism; D008208:Lymphatic System / Q000502:physiology*; D008297:Male; D008856:Microscopy, Fluorescence; D009130:Muscle, Smooth / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000096:biosynthesis; D052250:Nitric Oxide Synthase Type III; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000096:biosynthesis; D016133:Polymerase Chain Reaction; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D015534:Trans-Activators / Q000378:metabolism; D040321:Vascular Endothelial Growth Factor Receptor-3 / Q000096:biosynthesis; D033921:Vesicular Transport Proteins; D051176:beta Catenin
D000199:Actins; C495268:Ctnnb1 protein, rat; D003598:Cytoskeletal Proteins; D017931:DNA Primers; D006023:Glycoproteins; D016023:Integrins; C119248:LYVE1 protein, human; D008077:Lipoproteins, LDL; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D015534:Trans-Activators; D033921:Vesicular Transport Proteins; D051176:beta Catenin; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; C496316:Nos3 protein, rat; D040321:Vascular Endothelial Growth Factor Receptor-3