Ivabradine induces an atheroprotective gene expression profile in the endothelium of ApoE deficient mice before plaque formation

Atherosclerosis consists in a progressive accumulation of atheromatous plaques in medium and large caliber artery walls. Shear stress is generated by the friction of blood flow on the endothelium. Many studies have shown that plaques form in regions of the arterial tree, near bends and branches, characterized by low and oscillatory shear stress (disturbed), whereas linear regions exposed to unidirectional shear stress (laminar) are not susceptible to the plaques development. Hemodynamic studies have shown that the most atherogenic disturbed shear stress occurs in geometrically irregular regions during systolic phase, whereas, in the same regions, the magnitude of shear stress rises to less pathogenetic levels during diastole. The alteration of the disturbed shear stress during the cardiac cycle has a pivotal role in the context of atherosclerosis, since data from the literature indicate that an elevated heart rate stimulates the atherosclerotic plaques onset and progression. Ivabradine is a drug that reduces heart rate by selectively inhibiting the If current in the sinus node and numerous studies demonstrated that it limits atherosclerotic plaques formation in animal models. The mechanism of this protection is still unknown. It has been hypothesized that as heart rate decreases, following ivabradine treatment, the diastolic phase increases, thereby decreasing the exposure time of aortic arch endothelium to systolic flow, which is characterized by a more atherogenic low and oscillatory shear stress. The aim of this study was to determine the molecular effect of a short-term treatment with ivabradine in the initial steps of atherosclerosis development in an in vivo model of severe dyslipidemia (ApoE deficient-mouse), fed a standard diet. For this purpose, we performed a microarray analysis to determine the role of ivabradine in the modulation of gene expression in the endothelium of the aortic arch of ApoE -/- mice. Treatment induced changes in the expression of 930 transcripts. Shear stress-modulated MAPK signalling and sterol metabolic processes were among the most significantly affected pathways. We found upregulation of anti-inflammatory genes and downregulation of pro-apoptotic and pro-inflammatory genes, the majority of which were NF-kappaB- and/or AngII-regulated genes. Moreover, our results indicate that ivabradine modulates genes belonging to the Notch pathway, known to affect endothelial cells apoptosis and to be regulated by shear stress. Our in vitro flow experiments showed a sustained upregulation of Notch signalling by atheroprotective laminar shear stress. Our in vivo analysis on mice aorta confirms the activation of Notch signalling components by the atheroprotective laminar shear stress and provides the first in vivo evidence of differences in expression levels of Notch components between distinct regions of mouse aorta. In conclusion, this study provides strong evidence of ivabradine-mediated induction of a protective gene expression profile in the endothelium of aortic arch of ApoE-deficient mice. Among the identified pathways, Notch signalling modulation by ivabradine treatment may contribute this protection. Since numerous of these reported genes, including Notch, are shear stress modulated, our data are in agreement with the shared views that ivabradine, through heart rate reduction, modifies the shear stress on endothelium

Enhanced Patch-Clamp Technique to Study Antimicrobial Peptides and Viroporins, Inserted in a Cell Plasma Membrane with Fully Inactivated Endogenous Conductances

Many short peptides selectively permeabilize the bacteria plasma membrane, leading to their lyses and death: they are therefore a source of antibacterial molecules, and inspiration for novel and more selective drugs. Another class of short (<100 residues) membrane proteins called viroporins, because they are coded by viral genes, permeabilizes the membrane of susceptible cells during infection of by most animal viruses. The permeabilization leads to host cell lyses and the release of the virus mass, replicated at host cell expense, to propagate the infection. Detailed knowledge of the permeabilization properties of these proteins would allow to design, for instance, selective blockers of these pores, that would contrast the spread of the viral infection. In this chapter, the patch-clamp technique is employed to study the mechanism of membrane permeabilization induced by the pore-forming peptides, under strict physiological conditions. This goal is achieved by recording the ion current through the channels formed by these peptides, once inserted in a cell plasma membrane. To avoid contamination by the cell membrane currents, all the endogenous current sources must be blocked. It has been found that the photoreceptor rod outer segment mechanically isolated from the retina of low vertebrates (OS) was the most suitable cell to carry on the above studies, because it was possible to fully block all its endogenous currents without using any drug (such as TTX, TEA, dihydropyridines, etc.), that could obstruct the peptide pores or interfere with the pore formation. The peptides were applied to (and removed from) the extracellular OS side in ~50 ms with a computer-controlled microperfusion system, in which every perfusion parameter (as the rate of solution flow, the temporal sequence of solution changes or the number of automatic, self-washing cycles) was controlled by a user-friendly interface. This system allowed rapid application and removal of ions, drugs and peptides on the cells with a controlled timing, so that the ion channel characteristics (as its selectivity, blockade and gating) and the dynamics of pore formation could be precisely assessed. On the basis of the electrophysiological recordings obtained with representative peptides and with selected analogs, as alamethicin F50/5, the cecoprine-mellitin hybrid peptide, and a 20-aminoacid long fragment of the viroporin poliovirus 2B, it will be shown that the membrane pore formation occurs according to the barrel and stave, toroidal, and carpet model, respectively, that are the most widely-accepted mechanisms of membrane permeabilization. When recording large currents (produced for instance by high concentrations of peptides and/or highly permeable peptides), it is necessary to minimize series resistance, to reduce time constant of charging the cell membrane capacitance and error in membrane potential control. A second problem arises from the asymmetry of the plasma membrane: it is possible that the permeabilization properties of a particular peptide could be different depending upon the side of the membrane to which it is applied. For example, it is conceivable that viroporins are optimized to insert in the intracellular face of the plasma membrane, because they are synthesized in host cell cytosol. These two problems could be circumvented by widening the patch pipette shank, through the calibrated combination of heat and air pressure. These pipettes dramatically reduce series resistance, and allow at the same time to insert pulled quartz or plastic tubes very close to the pipette tip, making it possible the delivery of large molecules to the cytosol with a controlled timing. Finally, it is presented here a simple procedure to consistently attain seals with conventional or pressure polished pipettes, made from just one glass type, on a wide variety of cell types, isolated from different amphibian, reptilian, fish, and mammalian tissues, and on artificial membranes made with many different lipid mixtures

Notch Signaling Regulates Immune Responses in Atherosclerosis

Atherosclerosis is a chronic autoimmune inflammatory disease that can cause coronary artery disease, stroke, peripheral artery disease, depending on which arteries are affected. At the beginning of atherosclerosis plasma lipoproteins accumulate in the sub-endothelial space. In response, monocytes migrate from the circulation through the endothelium into the intima where they differentiate into macrophages. These early events trigger a complex immune response that eventually involves many cellular subtypes of both innate and adaptive immunity. The Notch signaling pathway is an evolutionary conserved cell signaling system that mediates cell-to-cell communication. Recent studies have revealed that Notch modulate atherosclerosis by controlling macrophages polarization into M1 or M2 subtypes. Furthermore, it is known that Notch signaling controls differentiation and activity of T-helper and cytotoxic T-cells in inflammatory diseases. In this review, we will discuss the role of Notch in modulating immunity in the context of atherosclerosis and whether targeting Notch may represent a therapeutic strategy

Nutraceuticals and Exercise against Muscle Wasting during Cancer Cachexia

Cancer cachexia (CC) is a debilitating multifactorial syndrome, involving progressive deterioration and functional impairment of skeletal muscles. It affects about 80% of patients with advanced cancer and causes premature death. No causal therapy is available against CC. In the last few decades, our understanding of the mechanisms contributing to muscle wasting during cancer has markedly increased. Both inflammation and oxidative stress (OS) alter anabolic and catabolic signaling pathways mostly culminating with muscle depletion. Several preclinical studies have emphasized the beneficial roles of several classes of nutraceuticals and modes of physical exercise, but their efficacy in CC patients remains scant. The route of nutraceutical administration is critical to increase its bioavailability and achieve the desired anti-cachexia effects. Accumulating evidence suggests that a single therapy may not be enough, and a bimodal intervention (nutraceuticals plus exercise) may be a more effective treatment for CC. This review focuses on the current state of the field on the role of inflammation and OS in the pathogenesis of muscle atrophy during CC, and how nutraceuticals and physical activity may act synergistically to limit muscle wasting and dysfunction

The Suppressor of AAC2 Lethality SAL1 Modulates Sensitivity of Heterologously Expressed Artemia ADP/ATP Carrier to Bongkrekate in Yeast

The ADP/ATP carrier protein (AAC) expressed in Artemia franciscana is refractory to bongkrekate. We generated two strains of Saccharomyces cerevisiae where AAC1 and AAC3 were inactivated and the AAC2 isoform was replaced with Artemia AAC containing a hemagglutinin tag (ArAAC-HA). In one of the strains the suppressor of ΔAAC2 lethality, SAL1, was also inactivated but a plasmid coding for yeast AAC2 was included, because the ArAACΔsal1Δ strain was lethal. In both strains ArAAC-HA was expressed and correctly localized to the mitochondria. Peptide sequencing of ArAAC expressed in Artemia and that expressed in the modified yeasts revealed identical amino acid sequences. The isolated mitochondria from both modified strains developed 85% of the membrane potential attained by mitochondria of control strains, and addition of ADP yielded bongkrekate-sensitive depolarizations implying acquired sensitivity of ArAAC-mediated adenine nucleotide exchange to this poison, independent from SAL1. However, growth of ArAAC-expressing yeasts in glycerol-containing media was arrested by bongkrekate only in the presence of SAL1. We conclude that the mitochondrial environment of yeasts relying on respiratory growth conferred sensitivity of ArAAC to bongkrekate in a SAL1-dependent manner. © 2013 Wysocka-Kapcinska et al

Divalent cations regulate pore formation of synthetic, naturally occurring alamethicin and selected analogs.

none4noneBenedusi M.; Milani A.; Aquila M.; Rispoli G.Benedusi, Mascia; Milani, Alberto; Aquila, Marco; Rispoli, Giorgi

Pressure-Polished Borosilicate Pipettes are “Universal Sealer” Yielding Low Access Resistance and Efficient Intracellular PerfusionPatch-Clamp Methods and Protocols

Whole-cell recording is the most widely used configuration of the patch recording technique, mainly because it allows to manipulate the intracellular environment while recording membrane current. However, the patch pipette tapered shank and the small tip opening give high access resistances and preclude efficient exchange between pipette solution and cell cytosol. Independently by the recording configuration, another problem of this technique is to gain consistently tight seals. Here it is described a method to enlarge the pipette shank without affecting the tip opening diameter, through the calibrated combination of heat and air pressure, with a custom made inexpensive set-up. These pressure polished pipettes give small access resistances, and allow the accommodation of pulled quartz or plastic perfusion tubes very close to the pipette tip (to deliver exogenous molecules into the cytosol with a controlled timing). Finally, it is also described a method to consistently attain seals with pipettes made from just one glass type, on a wide variety of cell types, isolated from different amphibian, reptilian, fish, and mammalian tissues, and on artificial membranes composed by many different lipid mixtures

Mechanism of pore formation of antimicrobial peptides and viroporins inserted in a cell plasma membrane

none4noneRispoli G; Benedusi M; Milani A; Aquila M.Rispoli, Giorgio; Benedusi, Mascia; Milani, Alberto; Aquila, Marc

Pore Forming Properties of Cecropin-Melittin Hybrid Peptide in a Natural Membrane

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