Isolation and characterization of a novel mouse lymphatic endothelial cell line: SV-LEC.
BACKGROUND
The lymphatic system regulates interstitial fluid and protein balance and modulates immune responses by regulating leukocyte and antigen traffic to lymph nodes. The present article describes a stable mouse lymphatic endothelial cell line from mesenteric adventitial tissue (SV-LEC) which is distinct from blood aortic (AEC) and venous (VEC) endothelial cells, based on expression of several lymphatic markers (e.g., Prox-1, LYVE-1, Flt-4). SV-LEC also expresses MAdCAM-1 in response to TNF-alpha, an effect seen in VEC, but not AEC.


RESULTS
Lymphatic endothelial cells (SV-LEC) were isolated from mesenteric adventitia from mice expressing temperature-sensitive SV40 large T ('Immortomouse', H-2K(b)tsA58) selected with hypoxia culture in D-valine-substituted MEM supplemented with VEGFC in a low oxygen atmosphere (0% O2, 5% CO2, and 95% N2) with 5 mM thioglycolate. Expression of lymphatic-specific markers (Flt-4, LYVE-1, Prox-1) and the tight junction proteins (ZO-1) were examined by RT-PCR, immunoblotting, and fluorescent microscopy. MAdCAM-1 (a high endothelial venular marker) expression was also examined in response to TNF-alpha IL-1beta and IFN-gamma.


RESULTS
Message for Flt-4 and LYVE-1 was detected on SV-LEC. Immunoblotting for LYVE-1 and Prox-1 showed strong expression on SV-LEC and VEC, but not AEC. Occludin expression was seen in all cell types, junctional ZO-1 was detected at SV-LEC and VEC junctions, not AEC.


CONCLUSIONS
SV-LEC expresses several lymphatic endothelial markers, some of which are shared with VEC, but not AEC, and may represent a useful system for modeling lymphatic function in vitro.
Ando
T
T
Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130-3932, USA.
Jordan
P
P
Joh
T
T
Wang
Y
Y
Jennings
M H
MH
Houghton
J
J
Alexander
J S
JS
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics / Q000378:metabolism; D015415:Biomarkers / Q000032:analysis*; D015152:Blotting, Northern; D015153:Blotting, Western; D015815:Cell Adhesion Molecules / Q000378:metabolism; D015687:Cell Hypoxia; D002460:Cell Line / Q000187:drug effects / Q000378:metabolism; D004729:Endothelium, Lymphatic / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism; D006720:Homozygote; D015151:Immunoblotting; D053583:Interleukin-1beta / Q000494:pharmacology; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins; D051379:Mice; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D008856:Microscopy, Fluorescence; D009088:Mucoproteins; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D040321:Vascular Endothelial Growth Factor Receptor-3 / Q000235:genetics / Q000378:metabolism; D062826:Zonula Occludens-1 Protein
D000952:Antigens, Polyomavirus Transforming; D015415:Biomarkers; D015815:Cell Adhesion Molecules; D006023:Glycoproteins; D053583:Interleukin-1beta; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009088:Mucoproteins; D010750:Phosphoproteins; D040321:Vascular Endothelial Growth Factor Receptor-3; D062826:Zonula Occludens-1 Protein; C489082:Madcam1 protein, mouse; C569043:Tjp1 protein, mouse; C492273:LYVE1 protein, mouse