Date of Award

12-1-2010

Degree Name

Doctor of Philosophy

Department

Molecular Biology, Microbiology and Biochemistry

First Advisor

Ran, Sophia

Abstract

Elevation of VEGFR-3, the primary mediator of lymphangiogenesis (i.e., new lymphatic vessel formation), is frequently associated with inflammation related to chronic disease and cancer. In the latter case, VEGFR-3 dependent lymphangiogenesis induced by inflamed tumors increases the incidence of distant metastasis, leading to decreased patient survival. However, the molecular mechanisms underlying inflammation-induced VEGFR-3 elevation and lymphangiogenesis are currently unknown. Two potential candidate genes that may regulate expression of VEGFR-3 are Prox1, the primary mediator of embryonic lymphangiogenesis, and NF-κB, the key intracellular regulator of inflammation-induced transcription. We hypothesized that the key inflammatory mediator, NF-κB, regulates transcription of key mediators of lymphangiogenesis, VEGFR-3 and Prox1. We further hypothesized that inflammation-induced elevation of VEGFR-3 and Prox1 are essential steps required for robust lymphangiogenesis in response to inflammation. The three primary goals of this study were to (1) delineate the time-course of events leading to inflammation-induced lymphangiogenesis in vivo; (2) clone and characterize the VEGFR-3 promoter and identify factors regulating VEGFR-3 expression in vitro; and (3) characterize the lymphatic phenotype of NF-κB p50 knockout mice. To begin testing these hypotheses, we used a mouse model of peritonitis to characterize induction of lymphangiogenesis and expression kinetics of NF-κB, Prox1 and VEGFR-3. In vivo time-course analysis of inflammation-induced lymphangiogenesis showed activation of NF-κB followed by sequential upregulation of Prox1 and VEGFR-3 that preceded lymphangiogenesis by 4 and 2 days, respectively. Characterization of the VEGFR-3 promoter by luciferase-reporter and ChIP assays showed direct activation by Prox1, NF-κB p50 and p65 transcription factors. This also revealed that Prox1 and NF-κB p50 bind in close proximity and synergistically activate the VEGFR-3 promoter. Characterization of p50 knockout mice revealed significantly decreased lymphatic vessel density in several organs that corresponded to reduced VEGFR-3 and Prox1 expression. Activation of NF-κB by inflammatory stimuli also elevated expression of NF-κB, Prox1 and VEGFR-3 in cultured lymphatic endothelial cells, which enhanced proliferation and migration in response to the VEGFR-3-specific ligand, VEGF-C152S. Collectively, our findings suggest that induction of the NF-κB pathway by inflammatory stimuli activates Prox1, and both NF-κB and Prox1 activate the VEGFR-3 promoter leading to increased receptor expression in lymphatic endothelial cells. This, in turn, enhances the responsiveness of pre-existing lymphatic endothelium to VEGFR-3 binding factors, VEGF-C and VEGF-D, ultimately resulting in robust lymphangiogenesis.

Share

COinS
 

Access

This dissertation is only available for download to the SIUC community. Others should contact the
interlibrary loan department of your local library or contact ProQuest's Dissertation Express service.