TNF induces distinct gene expression programs in microvascular and macrovascular human endothelial cells.

The relevance of the diversity of endothelial cells (ECs) for the response to inflammatory stimuli is currently not well defined. Using oligonucleotide microarray technique, we systematically analyzed the tumor necrosis factor (TNF)-induced expression profile in human microvascular ECs (HMEC) and macrovascular human umbilical vein ECs (HUVEC), analyzing 13,000 human genes by microarray analysis. Using strict inclusion and exclusion criteria, microarray analysis revealed that about half of the TNF-induced genes were specific for HMEC-1 or HUVEC. The microarray data could widely be confirmed by quantitative reverse transcriptase-polymerase chain reaction and at the protein level. It is interesting that the majority of those genes regulated depending on the cell type encoded for chemokines, cytokines, and cell surface molecules. Our results argue for a more careful consideration of specific effects restricted to distinct subtypes of ECs. The establishment of EC type-specific expression patterns may thus provide the basis for a selective manipulation of specific endothelial subtypes in different inflammatory diseases.

The contact allergen nickel triggers a unique inflammatory and proangiogenic gene expression pattern via activation of NF-kappaB and hypoxia-inducible factor-1alpha.

Nickel compounds are prime inducers of contact allergy reactions in humans. To identify the signal transduction pathways mediating the cellular responses to nickel and to elucidate their hierarchy, we performed Affymetrix gene profiling using human primary endothelial cells, which strongly respond to nickel stimulation. Overall, we found 258 significantly modulated transcripts, comprising 140 up-regulated and 118 down-regulated genes. The bulk of those genes were identified as targets of two distinct signaling cascades, the IKK2/NF-kappaB pathway and a proangiogenic pathway mediated by HIF-1alpha, which accumulates upon exposure to nickel. Using dominant-interfering mutants and retroviral RNA interference technology, we demonstrate that both pathways act independently to regulate expression of nonoverlapping gene pools. NF-kappaB activation mediates most of the proinflammatory responses to nickel. Nickel-dependent HIF-1alpha activation primarily modulates expression of genes involved in proliferation, survival, metabolism, and signaling, albeit the induction of some proinflammatory nickel-response genes, most prominently IL-6, which we identified as novel bona fide HIF-1alpha target in this study, is also critically dependent on this pathway. Furthermore, we provide evidence that transactivation of both transcription factors partially depends on p38 MAPK activation that contributes to the intensity of at least some target genes. Taken together, our data provide mechanistic insight into the complex network of nickel-induced cellular events and identify IKK2/NF-kappaB and HIF-1alpha as important pathways involved in processes such as delivery of "second signals" in contact hypersensitivity reactions to nickel.

NF{kappa}B-dependent down-regulation of tumor necrosis factor receptor-associated proteins contributes to interleukin-1-mediated enhancement of ultraviolet B-induced apoptosis.

Activation of the transcription factor nuclear factor-kappaB (NFkappaB) by inflammatory cytokines like tumor necrosis (TNF) factor and interleukin-1 (IL-1) is generally associated with the induction of antiapoptotic pathways. Therefore, NFkappaB inhibits both intrinsically and extrinsically induced apoptosis and thus is regarded to act universally in an antiapoptotic fashion. Accordingly, activation of NFkappaB by IL-1 was shown to result in reduction of death ligand-induced apoptosis via up-regulation of antiapoptotic inhibitor of apoptosis proteins (IAPs). In contrast, apoptosis induced by ultraviolet-B radiation (UVB) was shown to be enhanced in an NFkappaB-dependent manner, indicating that NFkappaB can also act in a proapoptotic fashion. This study investigates the molecular mechanisms underlying IL-1-mediated enhancement of UVB-induced apoptosis. We show that NFkappaB activation in costimulation with UVB treatment results in repression of antiapoptotic genes and consequently in down-regulation of the respective proteins, like c-IAP, FLICE-inhibitory protein (FLIP), and some members of the TNF receptor-associated (TRAF)2 protein family. In parallel, TNFalpha is released, leading to activation of signaling pathways mediated by TNF receptor-1 (TNF-R1). Although TNF is well known to induce both proapoptotic and antiapoptotic effects, the down-regulated levels of TRAF-1, -2, and -6 proteins by IL-1 plus UVB action leads to a shift toward promotion of the proapoptotic pathway. In concert with the down-regulation of IAPs and FLIP, TNF-R1 activation as an additional proapoptotic stimulus now results in significant enhancement of UVB-induced apoptosis. Taken together, elucidation of the molecular mechanisms underlying IL-1-mediated enhancement of UVB-induced apoptosis revealed that NFkappaB does not exclusively act in an antiapoptotic fashion but may also mediate proapoptotic effects.

Myeloid-related proteins 8 and 14 induce a specific inflammatory response in human microvascular endothelial cells.

Myeloid-related protein 8 (MRP8) and MRP14, S100 proteins secreted by activated phagocytes, bind specifically to endothelial cells. The endothelial response to MRP8/MRP14, however, is unknown. Using oligonucleotide microarray analysis, we show for the first time that MRP8/MRP14 induce a thrombogenic, inflammatory response in human microvascular endothelial cells by increasing the transcription of proinflammatory chemokines and adhesion molecules and by decreasing the expression of cell junction proteins and molecules involved in monolayer integrity. All changes on the gene expression level could be confirmed using biochemical and functional assays. We demonstrated that the expression of MRP8/MRP14 closely correlated with the inflammatory activity in systemic vasculitis, confirming the important role of these proteins for distinct inflammatory reactions in endothelia. MRP8/MRP14 may represent novel targets for anti-inflammatory strategies.

Transcriptional profiling of IKK2/NF-kappa B- and p38 MAP kinase-dependent gene expression in TNF-alpha-stimulated primary human endothelial cells.

Inflammatory stimulation of endothelial cells by tumor necrosis factor alpha (TNF-alpha) involves activation of nuclear factor kappa B (NF-kappa B) and p38 mitogen-activated protein (MAP) kinase signaling pathways. A reliable analysis of the gene expression program elicited by TNF-alpha and its assignment to distinct signaling pathways is not available. A sophisticated analysis of oligonucleotide microarrays covering more than 13 000 genes allowed definition of the TNF-alpha-regulated endothelial gene expression profile and novel TNF-alpha-induced genes. Virtually all TNF-alpha-inducible genes were dependent on I kappa B kinase 2 (IKK2)/NF-kappa B activation, whereas a minor number was additionally modulated by p38. Furthermore, genes suppressed by IKK2/NF-kappa B were newly identified. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and flow cytometry confirmed reliability of data. Thus, these results define a list of primary candidates for targeted modulation of endothelial functions during inflammation.