Endothelin-1 induces functionally active CD40 protein via nuclear factor-kappaB in human vascular smooth muscle cells.

A plethora of evidence supports a link between inflammation and atherogenesis. The vasoactive peptide endothelin-1 (ET-1) has both proatherogenic and proinflammatory properties. The CD40-CD154 signaling pathway exhibits a direct influence on atherogenesis. We therefore tested the hypothesis that ET-1 induces CD40 in human vascular smooth muscle cells (SMC). ET-1 concentration-dependently stimulated CD40 protein in SMC. The specific ET-A-receptor antagonist BQ-123 prevented CD40 induction demonstrating receptor specificity of the ET-1 effect. Experiments with PI-1, an inhibitor of the IkappaB-a-degrading proteasome complex, demonstrated involvement of the transcription factor NF-kappaB in ET-1-induced CD40 expression. Specific decoy oligodeoxynucleotides with the consensus binding sequence for NF-kappaB and AP-1 supported a NF-kappaB-dependent and AP-1-independent induction pathway. Functional relevance of ET-1-induced CD40 expression was demonstrated by an increase in IL-6 secretion after stimulation with CD154 of cells preactivated with ET-1. The data show a link between a proatherogenic vasoactive peptide and cell-cell contact mediated inflammatory pathways and may implicate novel therapeutic options for vascular disease.

Endothelin-1 induces CD40 but not IL-6 in human monocytes via the proinflammatory transcription factor NF-kappaB.

UNLABELLED: The vasoactive peptide endothelin-1 (ET-1) may contribute to the pathogenesis of atherosclerosis and its acute complications. Because inflammation of the vessel wall is a characteristic feature of atherosclerosis, this study investigated the effect of ET-1 on the proinflammatory transcription factor NF-kappaB in monocytes. Monocyte/macrophages are a major source of inflammatory mediators in atheroma and are located in rupture prone plaque areas. In human monocytes ET-1 caused NF-kappaB activation. Specificity of ET-1-induced NF-kappaB activation was ascertained by supershift and competition experiments. This ET-1 effect was blocked by the ET-A-receptor antagonist BQ-123 but not by the ET-B-receptor antagonist BQ-788. PI-1, a specific inhibitor of the IkappaB-alpha-degrading proteasome complex, also prevented NF-kappaB activation. ET-1 stimulated expression of the proinflammatory molecule CD40 but not of the cytokine IL-6 in a NF-kappaB-dependent manner. CONCLUSION: The data demonstrate the ability of ET-1 to activate inflammatory pathways in human monocytes differentially.

Endothelin-1 induces interleukin-6 release via activation of the transcription factor NF-kappaB in human vascular smooth muscle cells.

The potent vasoconstrictor peptide endothelin-1 (ET-1) has been implicated in the pathophysiology of atherosclerosis and its complications. Since inflammation of the vessel wall is a hallmark of atherosclerosis, the purpose of the present study was to investigate the influence of ET-1 on cytokine production in human vascular smooth muscle cells (SMC) as a marker of inflammatory cell activation. ET-1 (100 pM - 1 microM) stimulated interleukin-6 (IL-6) secretion from human vascular SMC in a concentration-dependent manner. The ET-A-receptor antagonist BQ-123 (10 microM), but not the ET-B-receptor antagonist BQ-788, inhibited IL-6 release. ET-1 also transiently increased IL-6 mRNA compatible with regulation of IL-6 release at the pretranslational level. Electrophoretic mobility shift assays demonstrated time- and concentration-dependent activation of the proinflammatory transcription factor nuclear factor-kappaB (NF-kappaB) in ET-1-stimulated human vascular SMC. A decoy oligodeoxynucleotide bearing the NF-kappaB binding site inhibited ET-1-stimulated IL-6 release to a great extent suggesting that this transcription factor plays a key role for cytokine production elicited by ET-1. Moreover, the antioxidant pyrrolidine dithiocarbamate (10 microM) inhibited ET-1-induced IL-6 release indicating involvement of reactive oxygen species in ET-1 signaling. ET-1-stimulated IL-6 secretion was also suppressed by diphenylene iodonium (40 microM), an inhibitor of flavon-containing enzymes such as NADH/NADPH oxidase. The results demonstrate the ability of ET-1 to induce an inflammatory response in human vascular SMC. These observations may contribute to a better understanding of the role of ET-1 in inflammatory activation of the vessel wall during atherogenesis.

Angiotensin II activates the proinflammatory transcription factor nuclear factor-kappaB in human monocytes.

The renin-angiotensin system may contribute to the pathogenesis of atherosclerosis. A common feature of all stages of atherosclerosis is inflammation of the vessel wall. The transcription factor nuclear factor-kappaB (NF-kappaB) participates in most signaling pathways involved in inflammation. This study therefore examined the effect of angiotensin (ANG) II on NF-kappaB activation in monocytic cells, a major cellular component of human atheroma, by electrophoretic mobility shift assay. ANG II, like TNFalpha, caused rapid activation of NF-kappaB in human mononuclear cells isolated from peripheral blood by Ficoll density gradient. This ANG II effect was blocked by the angiotensin AT1 receptor antagonist losartan. Specificity of ANG II-induced NF-kappaB activation was ascertained by supershift and competition experiments. Moreover, ANG II stimulated NF-kappaB activation in human monocytes, but not in lymphocytes from the same preparation. Together, the data demonstrate the ability of the vasoactive peptide ANG II to activate inflammatory pathways in human monocytes.

Human sodium channel myotonia: slowed channel inactivation due to substitutions for a glycine within the III-IV linker.

1. Three families with a form of myotonia (muscle stiffness due to membrane hyperexcitability) clinically distinct from previously classified myotonias were examined. The severity of the disease greatly differed among the families. 2. Three dominant point mutations were discovered at the same nucleotide position of the SCN4A gene encoding the adult skeletal muscle Na+ channel alpha-subunit. They predict the substitution of either glutamic acid, valine or alanine for glycine1306, a highly conserved residue within the supposed inactivation gate. Additional SCN4A mutations were excluded. 3. Electrophysiological studies were performed on biopsied muscle specimens obtained for each mutation. Patch clamp recordings on sarcolemmal blebs revealed an increase in the time constant of fast Na+ channel inactivation, tau h, and in late channel openings as compared to normal controls. tau h was increased from 1.2 to 1.6-2.1 ms and the average late currents from 0.4 to 1-6% of the peak early current. 4. Intracellular recordings on resealed fibre segments revealed an abnormal tetrodotoxin-sensitive steady-state inward current, and repetitive action potentials. Since K+ and Cl- conductances were normal, only the increase in the number of non-inactivating Na+ channels has to be responsible for the membrane hyperexcitability. 5. Length, ramification and charge of the side-chains of the substitutions correlated well with the Na+ channel dysfunction and the severity of myotonia, with alanine as the most benign and glutamic acid as the substitution with a major steric effect. 6. Our electrophysiological and molecular genetic studies strongly suggest that these Na+ channel mutations cause myotonia. The naturally occurring mutants allowed us to gain further insight into the mechanism of Na+ channel inactivation.