Abstract 3442: Inflammatory mediators activate lymphatic endothelial cells (LECs) through elevation of NF-κB, Prox1, and VEGFR-3
Authors: Flister Michael J. , Hall Kelly L. , Wilber Andrew , Ran Sophia 
Published: February 2011

Abstract
Introduction: Elevation of VEGFR-3, the primary mediator of lymphangiogenesis (i.e., new lymphatic vessel formation), is associated with chronically inflamed tumors and correlates with increased metastasis. However, the mechanisms regulating inflammation-induced expression of VEGFR-3 and lymphangiogenesis are unclear. Here we report that inflammatory mediators increase VEGFR-3 expression and activate cultured LECs. Moreover, inflammation-induced VEGFR-3 expression on new lymphatics in vivo was preceded by up-regulation of Prox1 and NF-κB, the key transcriptional regulators of lymphangiogenesis and inflammation, respectively.
Methods: Mouse peritonitis was induced by ip injection of LPS (100µg), thioglycollate (1.5%), or saline every 48h for 0-14 days. VEGFR-3, Prox1, and LYVE-1 expression and lymphatic vessel density were determined on diaphragms by IHC. VEGFR-3, Prox1, LYVE-1, NF-κB p50 and p65 expression was examined in cultured LECs treated with LPS (100ng/ml) or IL-3 (10ng/ml) for 6h and 24h. VEGFR-3 promoter transactivation by Prox1, p50 or p65 was measured by luciferase activity. ChIP analyses were performed using antibodies against Prox1, p50, p65, acetylated-H3, or non-specific IgG. Proliferation and migration of LECs induced by LPS, IL-3, and VEGF-C152S were examined by cell counting and modified Boyden's assay.
Results: Diaphragm lymphatic vessel density was significantly increased by two weeks of inflammation. This was preceded by increased Prox1 and phosphorylation of NF-κB p50 and p65 followed by up-regulation of VEGFR-3 expression on activated and newly-formed lymphatic vessels. In vitro, LPS and IL-3 increased VEGFR-3 expression in LECs by 6.2±0.8 and 4.4±0.7-fold, respectively. LPS and IL-3 also increased LEC proliferation and migration by 1.8 to 2.4-fold and 1.6 to 2.1-fold, correspondingly, and enhanced LEC activation by the VEGFR-3-specific ligand, VEGF-C152S by 20-40%. LEC exposure to LPS and IL-3 increased p50 and p65 of NF-κB by 2 to 3-fold. Prox1, NF-κB p50 and p65 activated the VEGFR-3−849/+55 promoter by 7-, 9-, and 1.8-fold, compared with VEGFR-3−436/−254 that was activated by 3-, 8-, and 2.2-fold, respectively. ChIP analysis confirmed binding of these transcription factors to the −403/−238bp segment of the VEGFR-3 promoter containing putative binding sites.
Conclusion: NF-κB-dependent inflammatory stimuli up-regulate Prox1 and VEGFR-3 expression in LECs. Promoter transactivation and ChIP assays suggest that Prox1 and NF-κB directly activate the proximal promoter leading to increased VEGFR-3 expression in response to inflammation. This, in turn, enhances responsiveness of pre-existing lymphatic endothelium to VEGFR-3 ligands resulting in robust lymphangiogenesis. These data suggest a central mechanism underlying tumor inflammation, lymphangiogenesis and metastasis, and present new targets for anti-metastatic therapy.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3442.