Abstract
Background: Endothelial dysfunction has been implicated in the pathogenesis of diverse pathologies ranging from vascular and immune diseases to cancer. TNF-alpha is one of the mediators of endothelial dysfunction through the activation of transcription factors, including NF-kappa B. While HUVEC (macrovascular cells) have been largely used in the past, here, we documented an NF-kappa B gene signature in TNF alpha-stimulated microvascular endothelial cells HMEC often used in tumor angiogenesis studies.
Methodology/Principal Findings: We measured mRNA expression of 55 NF-kappa B related genes using quantitative RT-PCR in HUVEC and HMEC. Our study identified twenty genes markedly up-regulated in response to TNF alpha, including adhesion molecules, cytokines, chemokines, and apoptosis regulators, some of them being identified as TNF-alpha-inducible genes for the first time in endothelial cells (two apoptosis regulators, TNFAIP3 and TNFRSF10B/Trail R2 (DR5), the chemokines GM-CSF/CSF2 and MCF/CSF1, and CD40 and TNF-alpha itself, as well as NF-kappa B components (RELB, NFKB1or 50/p105 and NFKB2 or p52/p100). For eight genes, the fold induction was much higher in HMEC, as compared to HUVEC. Most importantly, our study described for the first time a connection between NF-kappa B activation and the induction of most, if not all, of these genes in HMEC as evaluated by pharmacological inhibition and RelA expression knock-down by RNA interference. Moreover, since TNF-alpha is highly expressed in tumors, we further applied the NF-kappa B gene signature documented in TNF alpha-stimulated endothelial cells to human breast tumors. We found a significant positive correlation between TNF and the majority (85 %) of the identified endothelial TNF-induced genes in a well-defined series of 96 (48 ER alpha positive and 48 ER alpha negative) breast tumors.
Conclusion/Significance: Taken together these data suggest the potential use of this NF-kappa B gene signature in analyzing the role of TNF-alpha in the endothelial dysfunction, as well as in breast tumors independently of the presence of ER alpha.