Felipe Simon

Early lipopolysaccharide-induced reactive oxygen species production evokes necrotic cell death in human umbilical vein endothelial cells.

Background Endothelial dysfunction is a crucial step in the pathogenesis of cardiovascular diseases. Reactive oxygen species (ROS) generated in response to lipopolysaccharide (LPS) during sepsis promotes progressive endothelial failure. Typically, LPS-stimulated leukocytes produce pro-inflammatory cytokines, which trigger endothelial ROS production through NAD(P)H oxidase (Nox) activation, in a process that takes hours. Noteworthy, endothelial cells exposed to LPS may also generate ROS in just a few minutes. However, the mechanisms underlying this early event and its deleterious effect in endothelial function are unknown. Here, we investigated the mechanisms of early LPS-induced ROS generation and its effect in endothelial cell viability. Methods Human umbilical vein endothelial cells were exposed to LPS for 1–40 min to study ROS generation, cytokines expression, and signaling transduction by confocal microscopy, real-time PCR (RT-PCR), western blot, and immunoprecipation. Fourty-eight hour treatments were used to determine cell death by MTT assay, cell counting, and flow cytometry. Contribution of specific Nox isoform was evaluated using a siRNAs approach. Results LPS rapidly evoked a cytokine-independent ROS production, eliciting a rapid increase in p47phox phosphorylation by a phospholipase C/conventional protein kinase C and PI3-K signaling. It is noteworthy that the early LPS-induced ROS production triggered significant endothelial necrosis, which was prevented by a previous, but not a posterior, antioxidant treatment. The early LPS-induced ROS production as well as endothelial necrosis was totally dependent of Nox2 and Nox4 activity. Conclusion Endothelial cells exposure to LPS triggers an early ROS production. Remarkably, this single early ROS production is enough to generate extensive endothelial cell death by necrosis dependent on the activity of Nox2 and Nox4. Because, in sepsis, ROS production can cause endothelial dysfunction, results here provided may be relevant when considering the development of strategies for sepsis therapy.

Transient receptor potential melastatin 4 inhibition prevents lipopolysaccharide-induced endothelial cell death

AIMS Endothelial dysfunction is decisive in the progression of cardiovascular diseases. Lipopolysaccharide (LPS)-induced reactive oxygen species (ROS)-mediated endothelial cell death is a main feature observed in inflammation secondary to endotoxaemia, emerging as a leading cause of death among critically ill patients in intensive care units. However, the molecular mechanism underlying LPS-induced endothelial cell death is not well understood. Transient receptor protein melastatin 4 (TRPM4) is an ion channel associated with cell death that is expressed in endothelium and modulated by ROS. Here, we investigate the role of TRPM4 in LPS-induced endothelial cell death, testing whether suppression of the expression of TRPM4 confers endothelial cell resistance to LPS challenge. METHODS AND RESULTS Using primary cultures of human umbilical vein endothelial cells (HUVEC), we demonstrate that TRPM4 is critically involved in LPS-induced endothelial cell death. HUVEC exposed to LPS results in Na(+)-dependent cell death. Pharmacological inhibition of TRPM4 with 9-phenanthrol or glibenclamide protects endothelium against LPS exposure for 48 h. Furthermore, TRPM4-like currents increase in cells pre-treated with LPS and inhibited with glibenclamide. Of note, suppression of TRPM4 expression by siRNA or suppression of its activity in a dominant negative mutant is effective in decreasing LPS-induced endothelial cell death when cells are exposed to LPS for 24-30 h. CONCLUSION TRPM4 is critically involved in LPS-induced endothelial cell death. These results demonstrate that either pharmacological inhibition of TRPM4, suppression of TRPM4 expression, or inhibition of TRPM4 activity are able to protect endothelium against LPS injury. These results are useful in sepsis drug design and development of new strategies for sepsis therapy.

Angiotensin II-induced pro-fibrotic effects require p38MAPK activity and transforming growth factor beta 1 expression in skeletal muscle cells

Fibrotic disorders are typically characterised by excessive connective tissue and extracellular matrix (ECM) deposition that preclude the normal healing of different tissues. Several skeletal muscle dystrophies are characterised by extensive fibrosis. Among the factors involved in skeletal muscle fibrosis is angiotensin II (Ang-II), a key protein of the renin-angiotensin system (RAS). We previously demonstrated that myoblasts responded to Ang-II by increasing the ECM protein levels mediated by AT-1 receptors, implicating an Ang-II-induced reactive oxygen species (ROS) by a NAD(P)H oxidase-dependent mechanism. In this paper, we show that in myoblasts, Ang-II induced the increase of transforming growth factor beta 1 (TGF-β1) and connective tissue growth factor (CTGF) expression through its AT-1 receptor. This effect is dependent of the NAD(P)H oxidase (NOX)-induced ROS, as indicated by a decrease of the expression of both pro-fibrotic factors when the ROS production was inhibited via the NOX inhibitor apocynin. The increase in pro-fibrotic factors levels was paralleled by enhanced p38MAPK and ERK1/2 phosphorylation in response to Ang-II. However, only the p38MAPK activity was critical for the Ang-II-induced fibrotic effects, as indicated by the decrease in the Ang-II-induced TGF-β1 and CTGF expression and fibronectin levels by SB-203580, an inhibitor of the p38MAPK, but not by U0126, an inhibitor of ERK1/2 phosphorylation. Furthermore, we showed that the Ang-II-dependent p38MAPK activation, but not the ERK1/2 phosphorylation, was necessary for the NOX-derived ROS. In addition, we demonstrated that TGF-β1 expression was required for the Ang-II-induced pro-fibrotic effects evaluated by using SB-431542, an inhibitor of TGF-βRI kinase activity, and by knocking down TGF-β1 levels by shRNA technique. These results strongly suggest that the fibrotic response to Ang-II is mediated by the AT-1 receptor and requires the p38MAPK phosphorylation, NOX-induced ROS, and TGF-β1 expression increase mediated by Ang-II in skeletal muscle cells.

Fibrotic response induced by angiotensin-II requires NAD(P)H oxidase-induced reactive oxygen species (ROS) in skeletal muscle cells

Fibrotic disorders are typified by excessive connective tissue and extracellular matrix (ECM) deposition that precludes normal healing processes in different tissues. Angiotensin-II (Ang-II) is involved in the fibrotic response. Several muscular dystrophies are characterized by extensive fibrosis. However, the exact role of Ang-II in skeletal muscle fibrosis is unknown. Here we show that myoblasts responded to Ang-II by increasing protein levels of connective tissue growth factor (CTGF/CCN2), collagen-III and fibronectin. These Ang-II-induced pro-fibrotic effects were mediated by AT-1 receptors. Remarkably, Ang-II induced reactive oxygen species (ROS) via a NAD(P)H oxidase-dependent mechanism, as shown by inhibition of ROS production via the NAD(P)H oxidase inhibitors diphenylene iodonium (DPI) and apocynin. This increase in ROS is critical for Ang-II-induced fibrotic effects, as indicated by the decrease in Ang-II-induced CTGF and fibronectin levels by DPI and apocynin. We also show that Ang-II-induced ROS production and fibrosis require PKC activity as indicated by the generic PKC inhibitor chelerythrine. These results strongly suggest that the fibrotic response induced by Ang-II is mediated by AT-1 receptor and requires NAD(P)H-induced ROS in skeletal muscle cells.

Oxidative stress mediates the conversion of endothelial cells into myofibroblasts via a TGF-b1 and TGF-b2-dependent pathway

During the pathogenesis of systemic inflammation, reactive oxygen species (ROS) circulate in the bloodstream and interact with endothelial cells (ECs), increasing intracellular oxidative stress. Although endothelial dysfunction is crucial in the pathogenesis of systemic inflammation, little is known about the effects of oxidative stress on endothelial dysfunction. Oxidative stress induces several functions, including cellular transformation. A singular process of cell conversion is tendothelial-to-mesenchymal transition, in which ECs become myofibroblasts, thus losing their endothelial properties and gaining fibrotic behavior. However, the participation of oxidative stress as an inductor of conversion of ECs into myofibroblasts is not known. Thus, we studied the role played by oxidative stress in this conversion and investigated the underlying mechanism. Our results show that oxidative stress induces conversion of ECs into myofibroblasts through decreasing the levels of endothelial markers and increasing those of fibrotic and ECM proteins. The underlying mechanism depends on the ALK5/Smad3/NF-κB pathway. Oxidative stress induces the expression and secretion of TGF-β1 and TGF-β2 and p38 MAPK phosphorylation. Downregulation of TGF-β1 and TGF-β2 by siRNA technology abolished the H2O2-induced conversion. To our knowledge, this is the first report showing that oxidative stress is able to induce conversion of ECs into myofibroblasts via TGF-β secretion, emerging as a source for oxidative stress-based vascular dysfunction. Thus, oxidative stress emerges as a decisive factor in inducing conversion of ECs into myofibroblasts through a TGF-β-dependent mechanism, changing the ECs protein expression profile, and converting normal ECs into pathological ones. This information will be useful in designing new and improved therapeutic strategies against oxidative stress-mediated systemic inflammatory diseases.

Hydrogen Peroxide Removes TRPM4 Current Desensitization Conferring Increased Vulnerability to Necrotic Cell Death

Necrosis is associated with an increase in plasma membrane permeability, cell swelling, and loss of membrane integrity with subsequent release of cytoplasmic constituents. Severe redox imbalance by overproduction of reactive oxygen species is one of the main causes of necrosis. Here we demonstrate that H2O2 induces a sustained activity of TRPM4, a Ca2+-activated, Ca2+-impermeant nonselective cation channel resulting in an increased vulnerability to cell death. In HEK 293 cells overexpressing TRPM4, H2O2 was found to eliminate in a dose-dependent manner TRPM4 desensitization. Site-directed mutagenesis experiments revealed that the Cys1093 residue is crucial for the H2O2-mediated loss of desensitization. In HeLa cells, which endogenously express TRPM4, H2O2 elicited necrosis as well as apoptosis. H2O2-mediated necrosis but not apoptosis was abolished by replacement of external Na+ ions with sucrose or the non-permeant cation N-methyl-d-glucamine and by knocking down TRPM4 with a shRNA directed against TRPM4. Conversely, transient overexpression of TRPM4 in HeLa cells in which TRPM4 was previously silenced re-established vulnerability to H2O2-induced necrotic cell death. In addition, HeLa cells exposed to H2O2 displayed an irreversible loss of membrane potential, which was prevented by TRPM4 knockdown.

Role of astrocytes in memory and psychiatric disorders

Over the past decade, the traditional description of astrocytes as being merely accessories to brain function has shifted to one in which their role has been pushed into the forefront of importance. Current views suggest that astrocytes:(1) are excitable through calcium fluctuations and respond to neurotransmitters released at synapses; (2) communicate with each other via calcium waves and release their own gliotransmitters which are essential for synaptic plasticity; (3) activate hundreds of synapses at once, thereby synchronizing neuronal activity and activating or inhibiting complete neuronal networks; (4) release vasoactive substances to the smooth muscle surrounding blood vessels enabling the coupling of circulation (blood flow) to local brain activity; and (5) release lactate in an activity-dependent manner in order to supply neuronal metabolic demand. In consequence, the role of astrocytes and astrocytic gliotransmitters is now believed to be critical for higher brain function and recently, evidence begins to gather suggesting that astrocytes are pivotal for learning and memory. All of the above are reviewed here while focusing on the role of astrocytes in memory and psychiatric disorders.

Increased expression of the transient receptor potential melastatin 7 channel is critically involved in lipopolysaccharide-induced reactive oxygen species-mediated neuronal death

AIMS To assess the mechanisms involved in lipopolysaccharide (LPS)-induced neuronal cell death, we examined the cellular consequences of LPS exposure in differentiated PC12 neurons and primary hippocampal neurons. RESULTS Our data show that LPS is able to induce PC12 neuronal cell death without the participation of glial cells. Neuronal cell death was mediated by an increase in cellular reactive oxygen species (ROS) levels. Considering the prevalent role of specific ion channels in mediating the deleterious effect of ROS, we assessed their contribution to this process. Neurons exposed to LPS showed a significant intracellular Ca(2+) overload, and nonselective cationic channel blockers inhibited LPS-induced neuronal death. In particular, we observed that both LPS and hydrogen peroxide exposure strongly increased the expression of the transient receptor protein melastatin 7 (TRPM7), which is an ion channel directly implicated in neuronal cell death. Further, both LPS-induced TRPM7 overexpression and LPS-induced neuronal cell death were decreased with dithiothreitol, dipheniliodonium, and apocynin. Finally, knockdown of TRPM7 expression using small interference RNA technology protected primary hippocampal neurons and differentiated PC12 neurons from the LPS challenge. INNOVATION This is the first report showing that TRPM7 is a key protein involved in neuronal death after LPS challenge. CONCLUSION We conclude that LPS promotes an abnormal ROS-dependent TRPM7 overexpression, which plays a crucial role in pathologic events, thus leading to neuronal dysfunction and death.

Oxidative stress-modulated TRPM ion channels in cell dysfunction and pathological conditions in humans

The transient receptor potential melastatin (TRPM) protein family is an extensive group of ion channels expressed in several types of mammalian cells. Many studies have shown that these channels are crucial for performing several physiological functions. Additionally, a large body of evidence indicates that these channels are also involved in numerous human diseases, known as channelopathies. A characteristic event frequently observed during pathological states is the raising in intracellular oxidative agents over reducing molecules, shifting the redox balance and inducing oxidative stress. In particular, three members of the TRPM subfamily, TRPM2, TRPM4 and TRPM7, share the remarkable feature that their activities are modulated by oxidative stress. Because of the increase in oxidative stress, these TRPM channels function aberrantly, promoting the onset and development of diseases. Increases, absences, or modifications in the function of these redox-modulated TRPM channels are associated with cell dysfunction and human pathologies. Therefore, the effect of oxidative stress on ion channels becomes an essential part of the pathogenic mechanism. Thus, oxidative stress-modulated ion channels are more susceptible to generating pathological states than oxidant-independent channels. This review examines the most relevant findings regarding the participation of the oxidative stress-modulated TRPM ion channels, TRPM2, TRPM4, and TRPM7, in human diseases. In addition, the potential roles of these channels as therapeutic tools and targets for drug design are discussed.

Angiotensin-(1-7) decreases skeletal muscle atrophy induced by angiotensin II through a Mas receptor-dependent mechanism

Skeletal muscle atrophy is a pathological condition characterized by the loss of strength and muscle mass, an increase in myosin heavy chain (MHC) degradation and increase in the expression of two muscle-specific ubiquitin ligases: atrogin-1 and MuRF-1. Angiotensin II (AngII) induces muscle atrophy. Angiotensin-(1-7) [Ang-(1-7)], through its receptor Mas, produces the opposite effects than AngII. We assessed the effects of Ang-(1-7) on the skeletal muscle atrophy induced by AngII. Our results show that Ang-(1-7), through Mas, prevents the effects induced by AngII in muscle gastrocnemius: the decrease in the fibre diameter, muscle strength and MHC levels and the increase in atrogin-1 and MuRF-1. Ang-(1-7) also induces AKT phosphorylation. In addition, our analysis in vitro using C2C12 myotubes shows that Ang-(1-7), through a mechanism dependent on Mas, prevents the decrease in the levels of MHC and the increase in the expression of the atrogin-1 and MuRF-1, both induced by AngII. Ang-(1-7) induces AKT phosphorylation in myotubes; additionally, we demonstrated that the inhibition of AKT with MK-2206 decreases the anti-atrophic effects of Ang-(1-7). Thus, we demonstrate for the first time that Ang-(1-7) counteracts the skeletal muscle atrophy induced by AngII through a mechanism dependent on the Mas receptor, which involves AKT activity. Our study indicates that Ang-(1-7) is novel molecule with a potential therapeutical use to improve muscle wasting associated, at least, with pathologies that present high levels of AngII.