In vitrodifferentiation of human cardiac fibroblasts into myofibroblasts: characterization using electrical impedance.

Cardiac arrhythmias represent about 50% of the cardiovascular diseases which are the first cause of mortality in the world. Implantable medical devices play a major role for treating these arrhythmias. Nevertheless the leads induce an unwanted biological phenomenon called fibrosis. This phenomenon begins at a cellular level and is effective at a macroscopic scale causing tissue remodelling with a local modification of the active cardiac tissue. Fibrosis mechanism is complex but at the cellular level, it mainly consists in cardiac fibroblasts activation and differentiation into myofibroblasts. We developed a simplifiedin vitromodel of cardiac fibrosis, with human cardiac fibroblasts whom differentiation into myofibroblasts was promoted with TGF-ß1. Our study addresses an unreported impedance-based method for real-time monitoring ofin vitrocardiac fibrosis. The objective was to study whether the differentiation of cardiac fibroblasts in myofibroblasts had a specific signature on the cell index, an impedance-based feature measured by the xCELLigence system. Primary human cardiac fibroblasts were cultured along 6 days, with or without laminin coating, to study the role of this adhesion protein in cultures long-term maintenance. The cultures were characterized in the presence or absence of TGF-ß1 and we obtained a significant cell index signature specific to the human cardiac fibroblasts differentiation.

[Antiviral activities of interferon and PML pathway]. / Les mécanismes de l'action antivirale des interférons : la voie PML.

Discovered in 1957 for their antiviral properties, interferons (IFNs) are a growing cytokine family with diverse biological activities including antitumor and immunoregulatory activities. IFN are classified in three types I, II and III. They bind to different specific cell receptors and induce via the Jak/Stat pathway the expression of more than 300 genes, the products of which are believed to mediate their biological effects. Several proteins have been implicated in resistance to viral infection in IFN-treated cells, i.e. the dsRNAdependent protein kinase PKR, the 2'5' oligoadenylate synthetase/RNaseL and Mx proteins. However, it was demonstrated that cells from triple knockout mice lacking PKR, RNase L and Mx are still sensitive to the IFN-induced antiviral state, indicating that other pathways exist. One of these pathways implicates promyelocytic leukemia (PML) protein. This article reviews the potential antiviral activities of the different IFN-induced mediators focusing onPMLpathway and how viruses from different families overcome this defence.

Inducible NF-kappaB activation is permitted by simultaneous degradation of nuclear IkappaBalpha.

Signal-induced phosphorylation and ubiquitination of IkappaBalpha targets this inhibitor of NF-kappaB for proteasome-mediated degradation, thus permitting the release of active NF-kappaB. Upon cell stimulation, NF-kappaB activation results in neotranscription and neosynthesis of its own inhibitor, IkappaBalpha. As reported earlier, the neosynthesized inhibitor is then accumulated in the nucleus, where it rapidly binds to and terminates the function of nuclear NF-kappaB upon withdrawal of the stimulus. The present work was aimed at understanding how NF-kappaB activity is preserved while stimuli persist, despite intense, simultaneous IkappaBalpha neosynthesis, which would be expected to end NF-kappaB activity. We here show that incoming IkappaBalpha in the nucleus represents a target for resident nuclear proteasome complexes. Signal-induced, proteasome-dependent degradation of phosphorylated and ubiquitinated IkappaBalpha occurs in the nucleus, thus permitting the onset and persistence of NF-kappaB activity as long as stimulation is maintained. Our results suggest that intranuclear proteolysis of IkappaBalpha is necessarily required to avoid self-termination of NF-kappaB activity during cell activation.

Palmitoylation-dependent control of degradation, life span, and membrane expression of the CCR5 receptor.

We have shown that the chemokine and HIV receptor CCR5 is palmitoylated on a cluster of cysteine residues located at the boundary between the seventh transmembrane region and the cytoplasmic tail. Single or combined substitutions of the three cysteines (Cys-321, Cys-323, and Cys-324) or incubation of wild-type CCR5-transfected cells with the palmitic acid analog 2-bromopalmitate prevented palmitoylation of the receptor. Moreover, failure of CCR5 to be palmitoylated resulted in both accumulation in intracellular stores and a profound decrease of membrane expression of the receptor. Upon metabolic labeling, kinetic experiments showed that the half-life of palmitoylation-deficient CCR5 is profoundly decreased. Bafilomycin A1, but not a specific proteasome inhibitor, prevented early degradation of palmitoylation-deficient CCR5 and promoted its accumulation in lysosomal compartments. Although membrane expression of the CCR5 mutant was diminished, the molecules reaching the membrane were still able to interact efficiently with the chemokine ligand MIP1 beta and remained able to function as HIV co-receptors. Thus we conclude that palmitoylation controls CCR5 expression through regulation of the life span of this receptor.