Isolation and Characterization of A Novel Mouse Lymphatic Endothelial Cell Line: SV-LEC

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Abstract
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- α, an effect seen in VEC, but not AEC. Methods and Results: Lymphatic endothelial cells (SV-LEC) were isolated from mesenteric adventitia from mice expressing temperature-sensitive SV40 large T ('Immortomouse', H-2KbtsA58) 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- α IL-1 βand IFN- γ. 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. Conclusion: 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.