Gene expression profiling of HMGB1-stimulated endothelial cells: a balancing act between angiogenesis and inflammation

Aims HMGB1 (High Mobility Group box 1 or HMGB1) is a DNA-binding protein that was recently shown to have extracellular activities and play important roles in tumor metastasis, inflammation, and angiogenesis (1).This project aimed at the determination of the gene expression profile of Human endothelial cells stimulated with HMGB1 and evaluate its effects in an in vivo angiogenesis assay. Metodology Microarray analysis was performed using the Affimetrix Technology and the gene expression modulation was validated through Real time PCR. The CAM assay was performed using filter papers rings. Results The microarray data analysis on HUVEC cells stimulated with HMGB1 for 6 and 24 hours shows that HMGB1 significantly modulates genes involved in both angiogenesis and inflammation. After six hours of stimulation it upregulates a member of the vascular endothelial growth factor family but it downregulates Angiopoietin 2, a known vessel destabilizer that works in sinergism with VEGFA. At the same time point HMGB1 downregulates CXCR4, a receptor for the CXC family of cytokines (which, in concert with its ligand CXCL12 (SDF-1) is an angiogenic inducer), but it also downregulates CXCL11 which is a known anti-proliferative agent for endothelial cells. E-selectin, the chemokine IL-8 and the inflammatory cytokine TNF alpha are strongly upregulated. In vivo, in the chick allantoic membrane (CAM) assay, that we performed in presence of the anti inflammatory agent hydrocortisone, HMGB1 does not induce vascular growth while it reduces the angiogenesis induced by FGF-2. Conclusions There is a widely accepted causal link between inflammation and angiogenesis. Our data suggests a complex pattern in which, while HMGB1 is able to directly induce both pro- and anti-angiogenetic programs in endothelial cells, its pro-inflammatory activities on endothelial and other cell types, could in turn induce angiogenesis. To dissect at the molecular level the direct effects and mechanisms of action of HMGB1 in endothelial cells, we are investigating the role of the genes regulated by HMGB1 in modulating some typical phenotypes of angiogenic endothelial cells with both gain and loss of function (RNAi) approaches. Bibliography 1. Lotze MT, Tracey KJ. Nat Rev Immunol. 2005 Apr;5(4):331-42.